Processes for preparation of 9,11-epoxy steroids and intermediates useful therein

ABSTRACT

Multiple novel reaction schemes, novel process steps and novel intermediates are provided for the synthesis of epoxymexrenone and other compounds of Formula I                    
     wherein 
     —A—A— represents the group —CHR 4 —CHR 5 — or —CR 4 ═CR 5 —; 
     R 3 , R 4  and R 5  are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy; 
     R 1  represents an alpha-oriented lower alkoxycarbonyl or hydroxyalkyl radical; 
     —B—B— represents the group —CHR 6 —CHR 7 — or an alpha- or beta-oriented group:                    
     where R 6  and R 7  are independently selected from the group consisting of hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and 
     R 8  and R 9  are independently selected from the group consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy, or R 8  and R 9  together comprise a carbocyclic or heterocyclic ring structure, or R 8  or R 9  together with R 6  or comprise a carbocyclic or heterocyclic ring structure fused to the pentacyclic D ring.

This application is a continuation of U.S. application Ser. No.09/319,673 filed Dec. 13, 1999, now abandoned, which is a 371 ofPCT/US97/23090 filed Dec. 11, 1999, which claims priority to U.S.provisional Application Ser. No. 60/049,388 filed Jun. 11, 1997 and U.S.provisional Application Ser. No. 60/033,315 filed Dec. 11, 1996.

BACKGROUND OF THE INVENTION

This invention relates to the novel processes for the preparation of9,11-epoxy steroid compounds, especially those of the 20-spiroxaneseries and their analogs, novel intermediates useful in the preparationof steroid compounds, and processes for the preparation of such novelintermediates. Most particularly, the invention is directed to novel andadvantageous methods for the preparation of methyl hydrogen9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone(also referred to as eplerenone or epoxymexrenone).

Methods for the preparation of 20-spiroxane series compounds aredescribed in U.S. Pat. No. 4,559,332. The compounds produced inaccordance with the process of the '332 patent have an open oxygencontaining ring E of the general formula:

in which

—A—A— represents the group —CH₂—CH₂— or —CH═CH—;

R¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonylradical;

—B—B— represents the group —CH₂—CH₂— or an α- or β-oriented group;

R⁶ and R⁷ being hydrogen;

X represents two hydrogen atoms or oxo;

Y¹ and Y² together represent the oxygen bridge —O—, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy;

and salts of such compounds in which X represents oxo and Y² representshydroxy, that is to say of corresponding 17β-hydroxy-21-carboxylicacids.

U.S. Pat. No. 4,559,332 describes a number of methods for thepreparation of epoxymexrenone and related compounds of Formula IA. Theadvent of new and expanded clinical uses for epoxymexrenone create aneed for improved processes for the manufacture of this and otherrelated steroids.

SUMMARY OF THE INVENTION

The primary object of the present invention is the provision of improvedprocesses for the preparation of epoxymexrenone, other 20-spiroxanes andother steroids having common structural features. Among the particularobjects of the invention are: to provide an improved process thatproduces products of Formula IA and other related compounds in highyield; the provision of such a process which involves a minimum ofisolation steps; and the provision of such a process which may beimplemented with reasonable capital expense and operated at reasonableconversion cost.

Accordingly, the present invention is directed to a series of synthesisschemes for epoxymexrenone; intermediates useful in the manufacture ofepoxymexrenone; and syntheses for such novel intermediates.

The novel synthesis schemes are described in detail in the Descriptionof Preferred Embodiments. Among the novel intermediates of thisinvention are those described immediately below.

A compound of Formula IV corresponds to the structure:

wherein:

—A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—;

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxy carbonyl, cyano and aryloxy;

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical;

R² is an 11α-leaving group the abstraction of which is effective forgenerating a double bond between the 9- and 11-carbon atoms;

—B—B— represents the group —CHR⁶—CHR⁷— or an alpha- or beta-orientedgroup:

where

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy,or R⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

A compound of Formula IVA corresponds to Formula IV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula IVB corresponds to Formula IV wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae IVC, IVD and IVE, respectively, correspond to anyof Formula IV, IVA, or IVB wherein each of —A—A— and —B—B— is —CH₂—CH₂—,R³ is hydrogen, and R¹ is alkoxycarbonyl, preferably methoxycarbonyl.Compounds within the scope of Formula IV may be prepared by reacting alower alkylsulfonylating or acylating reagent, or a halide generatingagent, with a corresponding compound within the scope of Formula V.

A compound of Formula V corresponds to the structure:

wherein —A—A—, —B—B—, R¹, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VA corresponds to Formula V wherein R⁸ and R⁹ withthe ring carbon to which they are attached together form the structure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VB corresponds to Formula V wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae VC, VD and VE, respectively, correspond to any ofFormula V, VA, or VB wherein each of —A—A— and —B—B— is —CH₂—CH₂—, R³ ishydrogen, and R¹ is alkoxycarbonyl, preferably methoxycarbonyl.Compounds within the scope of Formula V may be prepared by reacting analkali metal alkoxide with a corresponding compound of Formula VI.

A compound of Formula VI corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIA corresponds to Formula VI wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIB corresponds to Formula VI wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae VIC, VID and VIE, respectively, correspond to anyof Formula VI, VIA, or VIB wherein each of —A—A— and —B—B— is —CH₂—CH₂—,and R³ is hydrogen. Compounds of Formula VI, VIA, VIB and VIC areprepared by hydrolyzing a compound corresponding to Formula VII, VIIA,VIIB or VIIC, respectively.

A compound of Formula VII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIIA corresponds to Formula VII wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIIB corresponds to Formula VII wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae VIIC, VIID and VIIE, respectively, correspond toany of Formula VII, VIIA, or VIIB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. A compound within the scope of FormulaVII may be prepared by cyanidation of a compound within the scope ofFormula VIII.

A compound of Formula VIII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIIIA corresponds to Formula VIII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIIIB corresponds to Formula VIII wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

Compounds of Formulae VIIIC, VIIID and VIIIE, respectively, correspondto any of Formula VIII, VIIIA, or VIIIB wherein each of —A—A— and —B—B—is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaVIII are prepared by oxidizing a substrate comprising a compound ofFormula XXX as described hereinbelow by fermentation effective forintroducing an 11-hydroxy group into the substrate in α-orientation.

A compound of Formula IX corresponds to the structure:

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV, and R¹is as defined in Formula V.

A compound of Formula IXA corresponds to Formula IX wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula IXB corresponds to Formula IX wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

Compounds of Formulae IXC, IXD and IXE, respectively, correspond to anyof Formula IX, IXA, or IXB wherein each of —A—A— and —B—B— is —CH₂—CH₂—,and R³ is hydrogen. Compounds within the scope of Formula IX can beprepared by bioconversion of a corresponding compound within the scopeof Formula X.

A compound of Formula XIV corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XIVA corresponds to Formula XIV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XIVB corresponds to Formula XIV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

Compounds of Formulae XIVC, XIVD and XIVE, respectively, correspond toany of Formula XIV, XIVA, or XIVB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of Formula XIVcan be prepared by hydrolysis of a corresponding compound within thescope of Formula XV.

A compound of Formula XV corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XVA corresponds to Formula XV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XVB corresponds to Formula XV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

Compounds of Formulae XVC, XVD and XVE, respectively, correspond to anyof Formula XV, XVA, or XVB wherein each of —A—A— and —B—B— is —CH₂—CH₂—,and R³ is hydrogen. Compounds within the scope of Formula XV can beprepared by cyanidation of a corresponding compound within the scope ofFormula XVI.

A compound of Formula XXI corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIA corresponds to Formula XXI wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIB corresponds to Formula XXI wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae XXIC, XXID and XXIE, respectively, correspond toany of Formula XXI, XXIA, or XXIB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of Formula XXImay be prepared by hydrolyzing a corresponding compound within the scopeof Formula XXII.

A compound of Formula XXII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIIA corresponds to Formula XXII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIIB corresponds to Formula XXII wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

Compounds of Formulae XXIIC, XXIID and XXIIE, respectively, correspondto any of Formula XXII, XXIIA, or XXIIB wherein each of —A—A— and —B—B—is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaXXII may be prepared by cyanidation of a compound within the scope ofFormula XXIII.

A compound of Formula XXIII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIIIA corresponds to Formula XXIII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIIIB corresponds to Formula XXIII wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

Compounds of Formulae XXIIIC, XXIIID and XXIIIE, respectively,correspond to any of Formula XXIII, XXIIIA, or XXIIIB wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula XXIII can be prepared by oxidation of a compound ofFormula XXIV, as described hereinbelow.

A compound of Formula XXVI corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXVIA corresponds to Formula XXVI wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula XXVI can be prepared by oxidation of a compound ofFormula XXVII.

A compound of Formula XXV corresponding to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXVA corresponds to Formula XXV wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula XXV can be prepared by cyanidation of a compound ofFormula XXVI.

A compound of Formula 104 corresponds to the structure:

wherein —A—A—, —B—B— and R³ are as defined in Formula IV, and R¹¹ is C₁to C₄ alkyl.

A compound of Formula 104A corresponds to Formula 104 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 104 may be prepared by thermal decomposition of acompound of Formula 103.

A compound of Formula 103 corresponds to the structure:

wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104, and R₁₂is a C₁ to C₄ alkyl.

A compound of Formula 103A corresponds to Formula 103 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 103 may be prepared by reaction of a correspondingcompound of Formula 102 with a dialkyl malonate in the presence of abase such as an alkali metal alkoxide.

A compound of Formula 102 corresponds to the structure:

wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 102A corresponds to Formula 102 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 102 may be prepared by reaction of a correspondingcompound of Formula 101 with a trialkyl sulfonium compound in thepresence of a base.

A compound of Formula 101 corresponds to the structure:

wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 101A corresponds to Formula 101 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 101 may be prepared by reaction of11α-hydroxyandrostene-3,17-dione or other compound of Formula XXXVI witha trialkyl orthoformate in the presence of an acid.

A compound of Formula XL corresponds to the Formula:

wherein —E—E— is selected from among:

R²¹, R²² and R²³ are independently selected from among hydrogen, alkyl,halo, nitro, and cyano; R²⁴ is selected from among hydrogen and loweralkyl; R⁸⁰ and R⁹⁰ are independently selected from keto and thesubstituents that may constitute R⁸ and R⁹ (as defined hereinabove withreference to Formula IV); and —A—A—, —B—B— and R³ are as defined inFormula IV.

A compound of Formula XLA corresponds to Formula XL wherein R²¹, R²² andR²³ are independently selected from among hydrogen, halogen and loweralkyl.

A compound of Formula XLB corresponds to Formula XLA wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII. A compound of FormulaXLC corresponds to Formula XLB wherein —E—E— corresponds to Formula XLV.A compound of XLD corresponds to Formula XLB wherein —E—E— correspondsto Formula XLVII.

A compound of Formula XLE corresponds to Formula XL wherein R⁸⁰ and R⁹⁰together with the ring carbon atom to which they are attached compriseketo or:

where X, Y¹, Y² and C(17) are as defined above, or

Compounds of Formula XLIE correspond to Formula XL in which R⁸⁰ and R⁹⁰together form keto.

Compounds of Formulae XLF, XLG, XLH, XLJ, XLM, and XLN correspond toFormula XL, XLA, XLB, XLC, XLD and XLE, respectively, in which —A—A—,—B—B— and R³ are as defined above.

A compound of Formula XLI corresponds to the Formula:

wherein —E—E— is selected from among:

R¹⁸ is C₁ to C₄ alkyl or the R¹⁸O-groups together form anO,O-oxyalkylene bridge; R²¹, R²² and R²³ are independently selected fromamong hydrogen, alkyl, halo, nitro, and cyano; R²⁴ is selected fromamong hydrogen and lower alkyl; R⁸⁰ and R⁹⁰ are independently selectedfrom keto and the substituents that may constitute R⁸ and R⁹; and —A—A—,—B—B— and R³ are as defined in Formula IV.

A compound of Formula XLIA corresponds to Formula XLI wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen, andlower alkyl.

A compound of Formula XLIB corresponds to Formula XLIA wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

A compound of Formula XLIC corresponds to Formula XLI wherein R⁸⁰ andR⁹⁰ together with the ring carbon atom to which they are attachedcomprise keto or:

where X, Y¹, Y² and C(17) are as defined above.

Compounds of Formulae XLID correspond to Formula XLI in which thesubstituent XXXIV corresponds to the structure XXXIII

Compounds of Formula XLIE correspond to Formula XL in which R⁸⁰ and R⁹⁰together form keto.

Compounds of Formulae XLIF, XLIG, XLIH, XLIJ, XLIM, and XLIN correspondto Formula XLI, XLIA, XLIB, XLIC, XLID and XLIE, respectively, in which—A—A—, —B—B— and R³ are as defined above. Compounds within the scope ofFormula XLI are prepared by hydrolysis of corresponding compounds ofFormula XL as defined hereinbelow.

A compound of Formula XLII corresponds to the Formula:

wherein —E—E— is selected from among:

R²¹, R²² and R²³ are independently selected from among hydrogen, alkyl,halo, nitro, and cyano; R²⁴ is selected from among hydrogen and loweralkyl; R⁸⁰ and R⁹⁰ are independently selected from keto and thesubstituents that may constitute R⁸ and R⁹; and —A—A—, —B—B— and R³ areas defined in Formula IV.

A compound of Formula XLIIA corresponds to Formula XLII wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen andlower alkyl.

A compound of Formula XLIIB corresponds to Formula XLIIA wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

A compound of Formula XLIIC corresponds to Formula XLII wherein R⁸⁰ andR⁹⁰ together with the ring carbon to which they are attached compriseketo or:

where X, Y¹, Y² and C(17) are as defined above.

Compounds of Formulae XLIID correspond to Formula XLII in which thesubstituent XXXIV corresponds to the structure XXXIII

Compounds of Formula XLIIE correspond to Formula XLII in which R⁸⁰ andR⁹⁰ together form keto. Compounds of Formulae XLIIF, XLIIG, XLIIH,XLIIJ, XLIIM and XLIIN correspond to Formulae XLII, XLIIA, XLIIB, XLIIC,XLIID and XLIIE, respectively, in which —A—A— and —B—B— are —CH₂—CH₂ andR³ is hydrogen. Compounds within the scope of Formula XLII are preparedby deprotecting a corresponding compound of Formula XLI.

A compound of the Formula XLIX corresponds to the structure:

wherein —E—E— is as defined in Formula XL, and —A—A—, —B—B—, R¹, R³, R⁸and R⁹ are as defined in Formula IV.

A compound of Formula XLIXA corresponds to Formula XLIX wherein R⁸ andR⁹ with the ring carbon to which they are attached together form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XLIXB corresponds to Formula XLIX wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

Compounds of Formulae XLIXC, XLIXD, XLIXE, respectively, correspond toany of Formula XLIX, XLIXA or XLIXB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, R³ is hydrogen and R¹ is alkoxycarbonyl, preferablymethoxycarbonyl. Compounds within the scope of Formula XLIX may beprepared by reacting an alcoholic or aqueous solvent with acorresponding compound Formula VI in the presence of a suitable base.

A compound of Formula A203 corresponds to the structure:

wherein —E—E— is selected from among:

R¹⁸ is selected from among C₁ to C₄ alkyl; R²¹, R²² and R²³ areindependently selected from among hydrogen, alkyl, halo, nitro, andcyano; R²⁴ is selected from among hydrogen and lower alkyl; and —A—A—,—B—B— and R³ are as defined in Formula IV.

A compound of Formula A203A corresponds to Formula A203 wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen, andlower alkyl.

A compound of Formula A203B corresponds to Formula A203A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

Compounds of Formulae A203C, A203D, and A203E respectively correspond toFormula A203, A203A and A203B wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaA203 are prepared by reducing a compound of Formula A202 as definedhereinbelow.

A compound of Formula A204 corresponds to the structure:

wherein R¹⁹ is C₁ to C₄ alkyl, and —E—E—, —A—A—, —B—B— and R³ are asdefined in Formula 203.

A compound of Formula A204A corresponds to Formula A204 wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen, andlower alkyl.

A compound of Formula A204B corresponds to Formula A204A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

Compounds of Formulae A204C, A204D, and A204E respectively correspond toFormulae A204, A204A, and A204B wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaA204 are prepared by hydrolysis of corresponding compounds of FormulaA203.

A compound of Formula A205 corresponds to the structure:

wherein R²⁰ is C₁ to C₄ alkyl, and —E—E—, R¹⁹, —A—A—, —B—B— and R³ areas defined in Formula 204.

A compound of Formula A205A corresponds to Formula A205 wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen, andlower alkyl.

A compound of Formula A205B corresponds to Formula A205A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

Compounds of Formulae A205C, A205D and A205E respectively correspond toFormula A205, A205A and A205B wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaA205 may be prepared by reacting a corresponding compound of FormulaA204 with an alkanol and acid.

A compound of Formula A206 corresponds to the structure:

wherein R¹⁹, R²⁰, —E—E—, —A—A—, —B—B— and R³ are as defined in Formula205.

A compound of Formula A206A corresponds to Formula A206 wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen, andlower alkyl.

A compound of Formula A206B corresponds to Formula A206A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

Compounds of Formulae A206C, A206D and A206E respectively correspond toFormula A206, A206A, and A206B wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaA206 may be prepared by reacting a corresponding compound within thescope of Formula A205 with a trialkyl sulfonium halide.

A compound of Formula A207 corresponds to the structure:

wherein R²⁵ is C₁ to C₄ alkyl, and —E—E—, R¹⁹, R²⁰, —A—A—, —B—B— and R³are as defined in Formula A205.

A compound of Formula A207A corresponds to Formula A207 wherein R²¹, R²²and R²³ are independently selected from among hydrogen, halogen, andlower alkyl.

A compound of Formula A207B corresponds to Formula A207A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

Compounds of Formulae A207C, A207D and A207E respectively correspond toFormula A207, A207A and A207B wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds of Formula A207 can be preparedby reaction of compounds of Formula A206 with a dialkyl malonate.

A compound of Formula A208 corresponds to the structure:

wherein —E—E—, R⁸⁰ and R⁹⁰ are as defined in Formula XLII; —A—A—, —B—B—and R³ are as defined in Formula 104; and R¹⁹, R²⁰, —A—A—, —B—B—, and R³are as defined in Formula 205.

A compound of Formula A208A corresponds to Formula A208 wherein R²¹ andR²² are independently selected from among hydrogen, halogen, and loweralkyl.

A compound of Formula A208B corresponds to Formula A208A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

A compound of Formula A208C corresponds to Formula A208 wherein R⁸⁰ andR⁹⁰ together with the ring carbon to which they are attached compriseketo or:

where X, Y¹, Y² and C(17) are as defined above.

Compounds of Formulae 208D correspond to Formula 208C in which thesubstituent XXXIV corresponds to the structure XXXIII

Compounds of Formulae A208E, A208F, A208G, A208H and A208J respectivelycorrespond to Formula A208, A208A, A208B, A208C and A208D wherein eachof —A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds withinthe scope of Formula A208 can be prepared by thermal decomposition ofcorresponding compounds of Formula A207.

A compound of Formula A209 corresponds to the structure:

wherein R⁸⁰ and R⁹⁰ are as defined in Formula XLI, and —E—E— and —A—A—,—B—B—, and R³ are as defined in Formula 205.

A compound of Formula A209A corresponds to Formula A209 wherein R²¹ andR²² are independently selected from among hydrogen, halogen, and loweralkyl.

A compound of Formula A209B corresponds to Formula A209A wherein —E—E—corresponds to Formula XLIII, XLIV, XLV or XLVII.

A compound of Formula A209C corresponds to Formula A209B wherein —E—E—corresponds to Formula XLIV.

A compound of Formula A209D corresponds to Formula A208 wherein R⁸⁰ andR⁹⁰ together with the ring carbon to which they are attached compriseketo or:

where X, Y¹, Y² and C(17) are as defined above.

Compounds of Formulae 209E correspond to Formula A209D in which thesubstituent XXXIV corresponds to the structure XXXIII

Compounds of Formulae A209F, A209G, A209H, A209J, A209L, and A209Mrespectively correspond to Formula A209, A209A, A209B, A209C, A209D andA209E wherein each of —A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen.Compounds within the scope of Formula A209 may be prepared by hydrolysisof a corresponding compound of Formula A208.

A compound of Formula A210 corresponds to the structure:

wherein R⁸⁰ and R⁹⁰ are as defined in Formula XLI, and the substituents—A—A—, —B—B— and R³ are as defined in Formula IV.

A compound of Formula A210A corresponds to Formula A210 wherein R⁸⁰ andR⁹⁰ together with the ring carbon to which they are attached compriseketo or:

wherein X, Y^(1, Y) ² and C(17) are as defined above.

Compounds of Formulae A210B correspond to Formula A210A in which thesubstituent XXXIV corresponds to the structure XXXIII

Compounds of Formula A210C correspond to Formula A210A in which R⁸⁰ andR⁹⁰ together form keto.

Compounds of Formulae A210D, A210E, A210F and A210G respectivelycorrespond to Formula A210, A210A, A210B and A210C wherein each of —A—A—and —B—B— is —CH₂—CH₂— and and R³ is hydrogen. Compounds within thescope of Formula 210 can be prepared by epoxidation of a compound ofFormula 209 in which —E—E— is

A compound of Formula A211 corresponds to the Formula

where —A—A—, —B—B— and R³ are as described above.

A compound of Formula A211A corresponds to Formula A211 wherein R⁸⁰ andR⁹⁰ together comprise keto or:

wherein X, Y¹, Y² and C(17) are as defined above.

Compounds of Formulae A211B correspond to Formula A211A in which thesubstituent XXXIV corresponds to the structure XXXIII

Compounds of Formula A211C correspond to Formula A210A in which R⁸⁰ andR⁹⁰ together form keto.

Compounds of Formulae A211D, A211E, A211F, and A211G, respectivelycorrespond to Formula A211, A211A, A211B and A211C wherein each of —A—A—and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scopeof Formula A211 can be prepared by oxidation of a corresponding compoundof Formula A210, or in the course of epoxidation of the correspondingcompound of Formula A209 where —E—E— is

Compounds of Formula A211 may be converted to compounds of Formula I inthe manner described hereinbelow.

A compound of Formula L corresponds to the structure:

wherein R¹¹ is C₁ to C₄ alkyl, and —A—A—, —B—B—, R¹, R², R³, R⁸ and R⁹are as defined above.

Compounds of Formula LA correspond to Formula L wherein R⁸ and R⁹together with the carbon atom to which they are attached comprises

wherein X, Y¹ and Y² are as defined above.

Compounds of Formula LB correspond to Formula L wherein R⁸ and R⁹correspond to Formula XXXIII

Compounds of Formulae LC, LD, LE correspond to Formulae L, LA and LB,respectively, where —A—A— and —B—B— are each —CH₂—CH₂— and R³ ishydrogen.

Based on the disclosure of specific reaction schemes as set outhereinbelow, it will be apparent which of these compounds have thegreatest utility relative to a particular reaction scheme. The compoundsof this invention are useful as intermediates for epoxymexrenone andother steroids.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow sheet of a process for the bioconversion ofcanrenone or a canrenone derivative to the corresponding 11α-hydroxycompound;

FIG. 2 is a schematic flow sheet of a preferred process for thebioconversion/11-α-hydroxylation of canrenone and canrenone derivatives;

FIG. 3 is a schematic flow sheet of a particularly preferred process forthe bioconversion/11-α-hydroxylation of canrenone and canrenonederivatives;

FIG. 4 shows the particle size distribution for canrenone as prepared inaccordance with the process of FIG. 2; and

FIG. 5 shows the particle size distribution for canrenone as sterilizedin the transformation fermenter in accordance with the process of FIG.3.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, various novel process schemeshave been devised for the preparation of epoxymexrenone and othercompounds corresponding Formula I:

wherein:

—A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—;

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxyalkylradical; and

—B—B— represents the group —CHR⁶—CHR⁷— or an alpha- or beta-orientedgroup:

where

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy,or R⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

Unless stated otherwise, organic radicals referred to as “lower” in thepresent disclosure contain at most 7, and preferably from 1 to 4, carbonatoms.

A lower alkoxycarbonyl radical is preferably one derived from an alkylradical having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec.-butyl and tert.-butyl; especiallypreferred are methoxycarbonyl, ethoxycarbonyl and isopropoxycarbonyl. Alower alkoxy radical is preferably one derived from one of theabove-mentioned C₁-C₄ alkyl radicals, especially from a primary C₁-C₄alkyl radical; especially preferred is methoxy. A lower alkanoyl radicalis preferably one derived from a straight-chain alkyl having from 1 to 7carbon atoms; especially preferred are formyl and acetyl.

A methylene bridge in the 15,16-position is preferably β-oriented.

A preferred class of compounds that may be produced in accordance withthe methods of the invention are the 20-spiroxane compounds described inU.S. Pat. No. 4,559,332, i.e., those corresponding to Formula IA:

where:

—A—A— represents the group —CH₂—CH₂— or —CH═CH—;

—B—B— represents the group —CH₂—CH₂— or an alpha- or beta-oriented groupof Formula IIIA:

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical;

X represents two hydrogen atoms, oxo or ═S;

Y¹ and Y² together represent the oxygen bridge —O—, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy.

Preferably, 20-spiroxane compounds produced by the novel methods of theinvention are those of Formula I in which Y¹ and Y² together representthe oxygen bridge —O—.

Especially preferred compounds of the formula I are those in which Xrepresents oxo. Of compounds of the 20-spiroxane compounds of Formula IAin which X represents oxo, there are most especially preferred those inwhich Y¹ together with Y² represents the oxygen bridge —O—.

As already mentioned, 17β-hydroxy-21-carboxylic acid may also be in theform of their salts. There come into consideration especially metal andammonium salts, such as alkali metal and alkaline earth metal salts, forexample sodium, calcium, magnesium and, preferably, potassium salts, andammonium salts derived from ammonia or a suitable, preferablyphysiologically tolerable, organic nitrogen-containing base. As basesthere come into consideration not only amines, for example loweralkylamines (such as triethylamine), hydroxy-lower alkylamines (such as2-hydroxyethylamine, di-(2-hydroxyethyl)-amine ortri-(2-hydroxyethyl)-amine), cycloalkylamines (such asdicyclohexylamine) or benzylamines (such as benzylamine andN,N′-dibenzylethylenediamine), but also nitrogen-containing heterocycliccompounds, for example those of aromatic character (such as pyridine orquinoline) or those having an at least partially saturated heterocyclicring (such as N-ethylpiperidine, morpholine, piperazine orN,N′-dimethylpiperazine).

Also included amongst preferred compounds are alkali metal salts,especially potassium salts, of compounds of the formula IA in which R¹represents alkoxycarbonyl, with X representing oxo and each of Y¹ and Y²representing hydroxy.

Especially preferred compounds of the formula I and IA are, for example,the following:

9α,11α-epoxy-7α-methoxycarbonyl-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-7α-ethoxycarbonyl-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-7α-isopropoxycarbonyl-20-spirox-4-ene-3,21-dione,

and the 1,2-dehydro analogue of each of the compounds;

9α,11α-epoxy-6α,7α-methylene-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-6β,7β-methylene-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-ene-3,21-dione,

and the 1,2-dehydro analogue of each of these compounds;

9α,11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-7α-isopropoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6α,7α-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6β,7β-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6β,7β;15β,16β-bismethylene-3-oxo-pregn-4-ene-21-carboxylicacid,

and alkali metal salts, especially the potassium salt or ammonium ofeach of these acids, and also a corresponding 1,2-dehydro analogue ofeach of the mentioned carboxylic acids or of a salt thereof;

9α,11α-epoxy-15β,16β-methylene-3,21-dioxo-20-spirox-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy-1565β,16β-methylene-3,21-dioxo-20-spiroxa-1,4-diene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy-3-oxo-20-spirox-4-ene-7α-carboxylic acid methyl ester,ethyl ester and isopropyl ester,

9α,11α-epoxy-6β,6β-methylene-20-spirox-4-en-3-one,

9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-en-3-one,

9α,11α-epoxy,17β-hydroxy-17α(3-hydroxy-propyl)-3-oxo-androst-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy,17β-hydroxy-17α-(3-hydroxypropyl)-6α,7α-methylene-androst-4-en-3-one,

9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6β,7β-methylene-androst-4-en-3-one,

9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6β,7β;15β,16β-bismethylene-androst-4-en-3-one,

including 17α-(3-acetoxypropyl) and 17α-(3-fromyloxypropyl) analogues ofthe mentioned androstane compounds,

and also 1,2-dehydro analogues of all the mentioned compounds of theandrost-4-en-3-one and 20-spirox-4-en-3-one series.

The chemical names of the compounds of the Formulae I and IA, and ofanalogue compounds having the same characteristic structural features,are derived according to current nomenclature in the following manner:for compounds in which Y¹ together with Y² represents —O—, from20-spiroxane (for example a compound of the formula IA in which Xrepresents oxo and Y¹ together with Y² represents —O— is derived from20-spiroxan-21-one); for those in which each of Y¹ and Y² representshydroxy and X represents oxo, from17β-hydroxy-17α-pregnene-21-carboxylic acid; and for those in which eachof Y¹ and Y² represents hydroxy and X represents two hydrogen atoms,from 17β-hydroxy-17α-(3-hydroxypropyl)-androstane. Since the cyclic andopen-chain forms, that is to say lactones and 17β-hydroxy-21-carboxylicacids and their salts, respectively, are so closely related to eachother that the latter may be considered merely as a hydrated form of theformer, there is to be understood hereinbefore and hereinafter, unlessspecifically stated otherwise, both in end products of the formula I andin starting materials and intermediates of analogous structure, in eachcase all the mentioned forms together.

In accordance with the invention, several separate process schemes havebeen devised for the preparation of compounds of Formula I in high yieldand at reasonable cost. Each of the synthesis schemes proceeds throughthe preparation of a series of intermediates. A number of theseintermediates are novel compounds, and the methods of preparation ofthese intermediates are novel processes.

Scheme 1 (Starting with Canrenone or Related Material)

One preferred process scheme for the preparation of compounds of FormulaI advantageously begins with canrenone or a related starting materialcorresponding to Formula XIII (or, alternatively, the process can beginwith androstendione or a related starting material)

wherein

—A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—;

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;

—B—B— represents the group —CHR⁶—CHR⁷— or an alpha- or beta-orientedgroup:

where

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy,or R⁸ and R⁹ together comprise a keto, carbocyclic or heterocyclic ringstructure, or R⁸ and R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

Using a bioconversion process of the type illustrated in FIGS. 1 and 2,an 11-hydroxy group of α-orientation is introduced in the compound ofFormula XIII, thereby producing a compound of Formula VIII:

where

—A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII. Preferably,the compound of Formula XIII has the structure

and the 11α-hydroxy product has the structure

in each of which

—A—A— represents the group —CH₂—CH₂— or —CH═CH—;

—B—B— represents the group —CH₂—CH₂— or an alpha- or beta-orientedgroup:

R³ is hydrogen, lower alkyl or lower alkoxy;

X represents two hydrogen atoms, oxo or ═S;

Y¹ and Y² together represent the oxygen bridge —O—, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy;

and salts of compounds in which X represents oxo and Y² representshydroxy. More preferably, the compound of Formula VIIIA produced in thereaction corresponds to a compound of Formula VIIIA wherein —A—A— and—B—B— are each —CH₂—CH₂—; R³ is hydrogen; Y¹, Y², and X are as definedin Formula XIIIA; and R⁸ and R⁹ together form the 20-spiroxanestructure:

Among the preferred organisms that can be used in this hydroxylationstep are Aspergillus ochraceus NRRL 405, Aspergillus ochraceus ATCC18500, Aspergillus niger ATCC 16888 and ATCC 26693, Aspergillus nidulansATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b,Streptomyces fradiae ATCC 10745, Bacillus megaterium ATCC 14945,Pseudomonas cruciviae ATCC 13262, and Trichothecium roseum ATCC 12543.Other preferred organisms include Fusarium oxysporum f.sp.cepae ATCC11171 and Rhizopus arrhizus ATCC 11145.

Other organisms that have exhibited activity for this reaction includeAbsidia coerula ATCC 6647, Absidia glauca ATCC 22752, Actinomucorelegans ATCC 6476, Aspergillus flavipes ATCC 1030, Aspergillus fumigatusATCC 26934, Beauveria bassiana ATCC 7159 and ATCC 13144, Botryosphaeriaobtusa IMI 038560, Calonectria decora ATCC 14767, Chaetomium cochliodesATCC 10195, Corynespora cassiicola ATCC 16718, Cunninghamellablakesleeana ATCC 8688a, Cunninghamella echinulata ATCC 3655,Cunninghamella elegans ATCC 9245, Curvularia clavata ATCC 22921,Curvularia lunata ACTT 12017, Cylindrocarpon radicicola ATCC 1011,Epicoccum humicola ATCC 12722, Gongronella butleri ATCC 22822, Hypomyceschrysospermus ATCC IMI 109891, Mortierella isabellina ATCC 42613, Mucormucedo ATCC 4605, Mucor qriseo-cyanus ATCC 1207A, Myrothecium verrucariaATCC 9095, Nocardia corallina ATCC 19070, Paecilomyces carneus ATCC46579, Penicillum patulum ATCC 24550, Pithomyces atro-olivaceus IFO6651, Pithomyces cynodontis ATCC 26150, Pycnosporium sp. ATCC 12231,Saccharopolyspora erythrae ATCC 11635, Sepedonium chrysospermum ATCC13378, Stachylidium bicolor ATCC 12672, Streptomyces hygroscopicus ATCC27438, Streptomyces purpurascens ATCC 25489, Syncephalastrum racemosumATCC 18192, Thamnostylum piriforme ATCC 8992, Thielavia terricola ATCC13807, and Verticillium theobromae ATCC 12474.

Additional organisms that may be expected to show activity for the11α-hydroxylation include Cephalosporium aphidicola (Phytochemistry(1996), 42(2), 411-415), Cochliobolus lunatas (J. Biotechnol. (1995),42(2), 145-150), Tieghemella orchidis (Khim.-Farm.Zh. (1986), 20(7),871-876), Tieghemella hyalospora (Khim.-Farm.Zh. (1986), 20(7),871-876), Monosporium olivaceum (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Aspergillus ustus (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Fusarium graminearum (Acta Microbiol. Pol., Ser. B.(1973), 5(2), 103-110), Verticillium glaucum (Acta Microbiol. Pol., Ser.B. (1973), 5(2), 103-110), and Rhizopus nigricans (J. Steroid Biochem.(1987), 28(2), 197-201).

The 11β-hydroxy derivatives of androstendione and mexrenone can beprepared according to the bioconversion processes set forth in Examples19A and 19B, respectively. The inventors hypothesize by analogy that thecorresponding β-hydroxy isomer of the compound of Formula VIII having aC11 β-hydroxy substituent instead of a C11 α-hydroxy substituent canalso be prepared using a similar bioconversion process employingsuitable microorganisms capable of carrying out the 11β-hydroxylation,such as one or more of the microorganisms disclosed herein.

Preparatory to production scale fermentation for hydroxylation ofcanrenone or other substrates of Formula XIII, an inoculum of cells isprepared in a seed fermentation system comprising a seed fermenter, or aseries of two or more seed fermenters. A working stock spore suspensionis introduced into the first seed fermenter, together with a nutrientsolution for growth of cells. If the volume of inoculum desired orneeded for production exceeds that produced in the first seed fermenter,the inoculum volume may be progressively and geometrically amplified byprogression through the remaining fermenters in the seed fermentationtrain. Preferably, the inoculum produced in the seed fermentation systemis of sufficient volume and viable cells for achieving rapid initiationof reaction in the production fermenter, relatively short productionbatch cycles, and high production fermenter activity. Whatever thenumber of vessels in a train of seed fermenters, the second andsubsequent seed fermenters are preferably sized so that the extent ofdilution at each step in the train is essentially the same. The initialdilution of inoculum in each seed fermenter can be approximately thesame as the dilution in the production fermenter. Canrenone or otherFormula XIII substrate is charged to the production fermenter along withinoculum and nutrient solution, and the hydroxylation reaction conductedthere.

The spore suspension charged to the seed fermentation system is from avial of working stock spore suspension taken from a plurality of vialsconstituting a working stock cell bank that is stored under cryogenicconditions prior to use. The working stock cell bank is in turn derivedfrom a master stock cell bank that has been prepared in the followingmanner. A spore specimen obtained from an appropriate source, e.g.,ATCC, is initially suspended in an aqueous medium such as, for example,saline solution, nutrient solution or a surfactant solution, (e.g., anonionic surfactant such as Tween 20 at a concentration of about 0.001%by weight), and the suspension distributed among culture plates, eachplate bearing a solid nutrient mixture, typically based on anon-digestible polysaccharide such as agar, where the spores arepropagated. The solid nutrient mixture preferably contains between about0.5% and about 5% by weight glucose, between about 0.05% and about 5% byweight of a nitrogen source, e.g., peptone, between about 0.05% andabout 0.5% by weight of a phosphorus source, e.g., an ammonium or alkalimetal phosphate such as dipotassium hydrogen phosphate, between about0.25% and about 2.5% by weight yeast lysate or extract (or other aminoacid source such as meat extract or brain heart infusion), between about1% and about 2% by weight agar or other non-digestible polysaccharide.Optionally, the solid nutrient mixture may further comprise and/orcontain between about 0.1% and about 5% by weight malt extract. The pHof the solid nutrient mixture is preferably between about 5.0 and about7.0, adjusted as required by alkali metal hydroxide or orthophosphoricacid. Among useful solid growth media are the following;

1. Solid Medium #1: 1% glucose, 0.25% yeast extract, 0.3% K₂HPO₄, and 2%agar (Bacto); pH adjusted to 6.5 with 20% NaOH. 2. Solid Medium #2: 2%peptone (Bacto), 1% yeast extract (Bacto), 2% glucose, and 2% agar(Bacto); pH adjusted to 5 with 10% H₃PO₄. 3. Solid Medium #3: 0.1%peptone (Bacto), 2% malt extract (Bacto), 2% glucose, and 2% agar(Bacto); pH as is 5.3. 4. Liquid Medium: 5% blackstrap molasses, 0.5%cornsteep liquor, 0.25% glucose, 0.25% NaCl, and 0.5% KH₂PO₄, pHadjusted to 5.8. 5. Difco Mycological agar (low pH).

The number of agar plates used in the development of a master stock cellbank can be selected with a view to future demands for master stock, buttypically about 15 to about 30 plates are so prepared. After a suitableperiod of growth, e.g., 7 to 10 days, the plates are scraped in thepresence of an aqueous vehicle, typically saline or buffer, forharvesting the spores, and the resulting master stock suspension isdivided among small vials, e.g., one ml. in each of a plurality of 1.5ml vials. To prepare a working stock spore suspension for use inresearch or production fermentation operations, the contents of one ormore of these second generation master stock vials can be distributedamong and incubated on agar plates in the manner described above for thepreparation of master stock spore suspension. Where routinemanufacturing operations are contemplated, as many as 100 to 400 platesmay be used to generate second generation working stock. Each plate isscraped into a separate working stock vial, each vial typicallycontaining one ml of the inoculum produced. For permanent preservation,both the master stock suspension and the second generation productioninoculum are advantageously stored in the vapor space of a cryogenicstorage vessel containing liquid N₂ or other cryogenic liquid.

In the process illustrated in FIG. 1, aqueous growth medium is preparedwhich includes a nitrogen source such as peptone, a yeast derivative orequivalent, glucose, and a source of phosphorus such as a phosphatesalt. Spores of the microorganism are cultured in this medium in theseed fermentation system. The preferred microorganism is Aspergillusochraceus NRRL 405 (ATCC 18500). The seed stock so produced is thenintroduced into the production fermenter together with the substrate ofFormula XIII. The fermentation broth is agitated and aerated for a timesufficient for the reaction to proceed to the desired degree ofcompletion.

The medium for the seed fermenter preferably comprises an aqueousmixture which contains: between about 0.5% and about 5% by weightglucose, between about 0.05% and about 5% by weight of a nitrogensource, e.g., peptone, between about 0.05% and about 0.5% by weight of aphosphorus source, e.g., an ammonium or alkali metal phosphate such asammonium phosphate monobasic or dipotassium hydrogen phosphate, betweenabout 0.25% and about 2.5% by weight yeast lysate or extract (or otheramino acid source such as distiller's solubles), between about 1% andabout 2% by weight agar or other non-digestable polysaccharide. Aparticularly preferred seed growth medium contains about 0.05% and about5% by weight of a nitrogen source such as peptone, between about 0.25%and about 2.5% by weight of autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose, and between about 0.05% byweight and about 0.5% by weight of a phosphorus source such as ammoniumphosphate monobasic. Especially economical process operations areafforded by the use of another preferred seed culture which containsbetween about 0.5% and about 5% by weight corn steep liquor, betweenabout 0.25% and about 2.5% autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose and about 0.05% and about 0.5%by weight ammonium phosphate monobasic. Corn steep liquor is aparticularly economical source of proteins, peptides, carbohydrates,organic acids, vitamins, metal ions, trace matters and phosphates. Mashliquors from other grains may be used in place of, or in addition to,corn steep liquor. The pH of the medium is preferably adjusted withinthe range of between about 5.0 and about 7.0, e.g., by addition of analkali metal hydroxide or orthophosphoric acid. Where corn steep liquorserves as the source of nitrogen and carbon, the pH is preferablyadjusted within the range of about 6.2 to about 6.8. The mediumcomprising peptone and glucose is preferably adjusted to a pH betweenabout 5.4 and about 6.2. Among useful growth media for use in seedfermentation:

1. Medium #1: 2% peptone, 2% yeast autolyzed (or yeast extract), and 2%glucose; pH adjusted to 5.8 with 20% NaOH. 2. Medium #2: 3% corn steepliquor, 1.5% yeast extract, 0.3% ammonium phosphate monobasic, and 3%glucose; pH adjusted to 6.5 with 20% NaOH.

Spores of the microorganism are introduced into this medium from a vialtypically containing in the neighborhood of 10⁹ spores per ml. ofsuspension. Optimal productivity of seed generation is realized wheredilution with growth medium at the beginning of a seed culture does notreduce the spore population density below about 10⁷ per ml. Preferably,the spores are cultured in the seed fermentation system until the packedmycelial volume (PMV) in the seed fermenter is at least about 20%,preferably about 35% to about 45%. Since the cycle in the seedfermentation vessel (or any vessel of a plurality which comprise a seedfermentation train) depends on the initial concentration in that vessel,it may be desirable to provide two or three seed fermentation stages toaccelerate the overall process. However, it is preferable to avoid theuse of significantly more than three seed fermenters in series, sinceactivity may be compromised if seed fermentation is carried through anexcessive number of stages. The seed culture fermentation is conductedunder agitation at a temperature in the range of about 23° to about 37°C., preferably in range of between about 24° and about 28° C.

Culture from the seed fermentation system is introduced into aproduction fermenter, together with a production growth medium. In oneembodiment of the invention, non-sterile canrenone or other substrate ofFormula XIII serves as the substrate for the reaction. Preferably, thesubstrate is added to the production fermenter in the form of a 10% to30% by weight slurry in growth medium. To increase the surface areaavailable for 11α-hydroxylation reaction, the particle size of theFormula XIII substrate is reduced by passing the substrate through anoff line micronizer prior to introduction into the fermenter. A sterilenutrient feed stock containing glucose, and a second sterile nutrientsolution containing a yeast derivative such as autolyzed yeast (orequivalent amino acid formulation based on alternative sources such asdistiller's solubles), are also separately introduced. The mediumcomprises an aqueous mixture containing: between about 0.5% and about 5%by weight glucose, between about 0.05% and about 5% by weight of anitrogen source, e.g., peptone, between about 0.05% and about 0.5% byweight of a phosphorus source, e.g., an ammonium or alkali metalphosphate such as dipotassium hydrogen phosphate, between about 0.25%and about 2.5% by weight yeast lysate or extract (or other amino acidsource such as distiller's solubles), between about 1% and about 2% byweight agar or other non-digestible polysaccharide. A particularlypreferred production growth medium contains about 0.05% and about 5% byweight of a nitrogen source such as peptone, between about 0.25% andabout 2.5% by weight of autolyzed yeast or yeast extract, between about0.5% and about 5% by weight glucose, and between about 0.05% and about0.5% by weight of a phosphorus source such as ammonium phosphatemonobasic. Another preferred production medium contains between about0.5% and about 5% by weight corn steep liquor, between about 0.25% andabout 2.5% autolyzed yeast or yeast extract, between about 0.5% andabout 5% by weight glucose and about 0.05% and about 0.5% by weightammonium phosphate monobasic. The pH of the production fermentationmedium is preferably adjusted in the manner described above for the seedfermentation medium, with the same preferred ranges for the pH ofpeptone/glucose based media and corn steep liquor based media,respectively. Useful bioconversion growth media are set forth below:

1. Medium #1: 2% peptone, 2% yeast autolyzed (or yeast extract), and 2%glucose; pH adjusted to 5.8 with 20% NaOH. 2. Medium #2: 1% peptone, 1%yeast autolyzed (or yeast extract), and 2% glucose; pH adjusted to 5.8with 20% NaOH. 3. Medium #3: 0.5% peptone, 0.5% yeast autolyzed (oryeast extract), and 0.5% glucose; pH adjusted to 5.8 with 20% NaOH. 4.Medium #4: 3% corn steep liquor, 1.5% yeast extract, 0.3% ammoniumphosphate monobasic, and 3% glucose; pH adjusted to 6.5 with 20% NaOH.5. Medium #5: 2.55% corn steep liquor, 1.275% yeast extract, 0.255%ammonium phosphate monobasic, and 3% glucose; pH adjusted to 6.5 with20% NaOH. 6. Medium #6: 2.1% corn steep liquor, 1.05% yeast extract,0.21% ammonium phosphate monobasic, and 3% glucose; pH adjusted to 6.5with 20% NaOH.

Non-sterile canrenone and sterile nutrient solutions are chain fed tothe production fermenter in about five to about twenty, preferably aboutten to about fifteen, preferably substantially equal, portions each overthe production batch cycle. Advantageously, the substrate is initiallyintroduced in an amount sufficient to establish a concentration ofbetween about 0.1% by weight and about 3% by weight, preferably betweenabout 0.5% and about 2% by weight, before inoculation with seedfermentation broth, then added periodically, conveniently every 8 to 24hours, to a cumulative proportion of between about 1% and about 8% byweight. Where additional substrate is added every 8 hour shift, totaladdition may be slightly lower, e.g., 0.25% to 2.5% by weight, than inthe case where substrate is added only on a daily basis. In the latterinstance cumulative canrenone addition may need to be in the range 2% toabout 8% by weight. The supplemental nutrient mixture fed during thefermentation reaction is preferably a concentrate, for example, amixture containing between about 40% and about 60% by weight sterileglucose, and between about 16% and about 32% by weight sterile yeastextract or other sterile source of yeast derivative (or other amino acidsource). Since the substrate fed to the production fermenter of FIG. 1is non-sterile, antibiotics are periodically added to the fermentationbroth to control the growth of undesired organisms. Antibiotics such askanamycin, tetracycline, and cefalexin can be added withoutdisadvantageously affecting growth and bioconversion. Preferably, theseare introduced into the fermentation broth in a concentration, e.g., ofbetween about 0.0004% and about 0.002% based on the total amount of thebroth, comprising, e.g., between about 0.0002% and about 0.0006%kanamycin sulfate, between about 0.0002% and about 0.006% tetracyclineHCl and/or between about 0.001% and about 0.003% cefalexin, again basedon the total amount of broth.

Typically, the production fermentation batch cycle is in theneighborhood of about 80-160 hours. Thus, portions of each of theFormula XIII substrates and nutrient solutions are typically added aboutevery 2 to 10 hours, preferably about every 4 to 6 hours.Advantageously, an antifoam is also incorporated in the seedfermentation system, and in the production fermenter.

Preferably, in the process of FIG. 1, the inoculum charge to theproduction fermenter is about 0.5% to about 7%, more preferably about 1%to about 2%, by volume based on the total mixture in the fermenter, andthe glucose concentration is maintained between about 0.01% and about1.0%, preferably between about 0.025% and about 0.5%, more preferablybetween about 0.05% and about 0.25% by weight with periodic additionsthat are preferably in portions of about 0.05% to about 0.25% by weight,based on the total batch charge. The fermentation temperature isconveniently controlled within a range of about 20° to about 37° C.,preferably about 24° C. to about 28° C., but it may be desirable to stepdown the temperature during the reaction, e.g., in 2° C. increments, tomaintain the packed mycelium volume (PMV) below about 60%, morepreferably below about 50%, and thereby prevent the viscosity of thefermentation broth from interfering with satisfactory mixing. If thebiomass growth extends above the liquid surface, substrate retainedwithin the biomass may be carried out of the reaction zone and becomeunavailable for the hydroxylation reaction. For productivity, it isdesirable to reach a PMV in the range of 30 to 50%, preferably 35% to45%, within the first 24 hours of the fermentation reaction, butthereafter conditions are preferably managed to control further growthwithin the limits stated above. During reaction, the pH of thefermentation medium is controlled at between about 5.0 and about 6.5,preferably between about 5.2 and about 5.8, and the fermenter isagitated at a rate of between about 400 and about 800 rpm. A dissolvedoxygen level of at least about 10% of saturation is achieved by aeratingthe batch at between about 0.2 and about 1.0 vvm, and maintaining thepressure in the head space of the fermenter at between about atmosphericand about 1.0 bar gauge, most preferably in the neighborhood of about0.7 bar gauge. Agitation rate may also be increased as necessary tomaintain minimum dissolved oxygen levels. Advantageously, the dissolvedoxygen is maintained at well above about 10%, in fact as high as about50% to promote conversion of substrate. Maintaining the pH in the rangeof 5.5±0.2 is also optimal for bioconversion. Foaming is controlled asnecessary by addition of a common antifoaming agent. After all substratehas been added, reaction is preferably continued until the molar ratioof Formula VIII product to remaining unreacted Formula XIII substrate isat least about 9 to 1. Such conversion may be achieve within the 80-160hour batch cycle indicated above.

It has been found that high conversions are associated with depletion ofinitial nutrient levels below the initial charge level, and bycontrolling aeration rate and agitation rate to avoid splashing ofsubstrate out of the liquid broth. In the process of FIG. 1, thenutrient level was depleted to and then maintained at no greater thanabout 60%, preferably about 50%, of the initial charge level; while inthe processes of FIGS. 2 and 3, the nutrient level was reduced to andmaintained at no greater than about 80%, preferably about 70%, of theinitial charge level. Aeration rate is preferably no greater than onevvm, more preferably in the range of about 0.5 vvm; while agitation rateis preferably not greater than 600 rpm.

A particularly preferred process for preparation of a compound ofFormula VIII is illustrated in FIG. 2. A preferred microorganism for the11α-hydroxylation of a compound of Formula XIII (for example, canrenone)is Aspergillus ochraceus NRRL 405 (ATCC 18500). In this process, growthmedium preferably comprises between about 0.5% and about 5% by weightcorn steep liquor, between about 0.5% and about 5% by weight glucose,between about 0.1% and about 3% by weight yeast extract, and betweenabout 0.05% and about 0.5% by weight ammonium phosphate. However, otherproduction growth media as described herein may also be used. The seedculture is prepared essentially in the manner described for the processof FIG. 1, using any of the seed fermentation media described herein. Asuspension of non-micronized canrenone or other Formula XIII substratein the growth medium is prepared aseptically in a blender, preferably ata relatively high concentration of between about 10% and about 30% byweight substrate. Preferably, aseptic preparation may comprisesterilization or pasteurization of the suspension after mixing. Theentire amount of sterile substrate suspension required for a productionbatch is introduced into the production fermenter at the beginning ofthe batch, or by periodical chain feeding. The particle size of thesubstrate is reduced by wet milling in an on-line shear pump whichtransfers the slurry to the production fermenter, thus obviating theneed for use of an off-line micronizer. Where aseptic conditions areachieved by pasteurization rather than sterilization, the extent ofagglomeration may be insignificant, but the use of a shear pump may bedesirable to provide positive control of particle size. Sterile growthmedium and glucose solution are introduced into the production fermenteressentially in the same manner as described above. All feed componentsto the production fermenter are sterilized before introduction, so thatno antibiotics are required.

Preferably, in operation of the process of FIG. 2, the inoculum isintroduced into the production fermenter in a proportion of betweenabout 0.5% and about 7%, the fermentation temperature is between about20° and about 37° C., preferably between about 24° C. and about 28° C.,and the pH is controlled between about 4.4 and about 6.5, preferablybetween about 5.3 and about 5.5, e.g., by introduction of gaseousammonia, aqueous ammonium hydroxide, aqueous alkali metal hydroxide, ororthophosphoric acid. As in the process of FIG. 1, the temperature ispreferably trimmed to control growth of the biomass so that PMV does notexceed 55-60%. The initial glucose charge is preferably between about 1%and about 4% by weight, most preferably 2.5% to 3.5% by weight, but ispreferably allowed to drift below about 1.0% by weight duringfermentation. Supplemental glucose is fed periodically in portions ofbetween about 0.2% and about 1.0% by weight based on the total batchcharge, so as to maintain the glucose concentration in the fermentationzone within a range of between about 0.1% and about 1.5% by weight,preferably between about 0.25% and about 0.5% by weight. Optionally,nitrogen and phosphorus sources may be supplemented along with glucose.However, because the entire canrenone charge is made at the beginning ofthe batch cycle, the requisite supply of nitrogen and phosphorus bearingnutrients can also be introduced at that time, allowing the use of onlya glucose solution for supplementation during the reaction. The rate andnature of agitation is a significant variable. Moderately vigorousagitation promotes mass transfer between the solid substrate and theaqueous phase. However, a low shear impeller should be used to preventdegradation of the myelin of the microorganisms. Optimal agitationvelocity varies within the range of 200 to 800 rpm, depending on culturebroth viscosity, oxygen concentration, and mixing conditions as affectedby vessel, baffle and impeller configuration. Ordinarily, a preferredagitation rate is in the range of 350-600 rpm. Preferably the agitationimpeller provides a downward axially pumping function so as to assist ingood mixing of the fermented biomass. The batch is preferably aerated ata rate of between about 0.3 and about 1.0 vvm, preferably 0.4 to 0.8vvm, and the pressure in the head space of the fermenter is preferablybetween about 0.5 and about 1.0 bar gauge. Temperature, agitation,aeration and back pressure are preferably controlled to maintaindissolved oxygen in the range of at least about 10% by volume during thebioconversion. Total batch cycle is typically between about 100 andabout 140 hours.

Although the principle of operation for the process of FIG. 2 is basedon early introduction of substantially the entire canrenone charge, itwill be understood that growth of the fermentation broth may be carriedout before the bulk of the canrenone is charged. Optionally, someportion of the canrenone can also be added later in the batch.Generally, however, at least about 75% of the sterile canrenone chargeshould be introduced into the transformation fermenter within 48 hoursafter initiation of fermentation. Moreover, it is desirable to introduceat least about 25% by weight canrenone at the beginning of thefermentation, or at least within the first 24 hours in order to promotegeneration of the bioconversion enzyme(s).

In a further preferred process as illustrated in FIG. 3, the entirebatch charge and nutrient solution are sterilized in the productionfermentation vessel prior to the introduction of inoculum. The nutrientsolutions that may be used, as well as the preferences among them, areessentially the same as in the process of FIG. 2. In this embodiment ofthe invention, the shearing action of the agitator impeller breaks downthe substrate agglomerates that otherwise tend to form uponsterilization. It has been found that the reaction proceedssatisfactorily if the mean particle size of the canrenone is less thanabout 300μ and at least 75% by weight of the particles are smaller than240μ. The use of a suitable impeller, e.g., a disk turbine impeller, atan adequate velocity in the range of 200 to 800 rpm, with a tip speed ofat least about 400 cm/sec., has been found to provide a shear ratesufficient to maintain such particle size characteristics despite theagglomeration that tends to occur upon sterilization within theproduction fermenter. The remaining operation of the process of FIG. 3is essentially the same as the process of FIG. 2. The processes of FIGS.2 and 3 offer several distinct advantages over the process of FIG. 1. Aparticular advantage is the amenability to use of a low cost nutrientbase such as corn steep liquor. But further advantages are realized ineliminating the need of antibiotics, simplifying feeding procedures, andallowing for batch sterilization of canrenone or other Formula XIIIsubstrate. Another particular advantage is the ability to use a simpleglucose solution rather than a complex nutrient solution forsupplementation during the reaction cycle.

In processes depicted in FIGS. 1 to 3, the product of Formula VIII is acrystalline solid which, together with the biomass, may be separatedfrom the reaction broth by filtration or low speed centrifugation.Alternatively, the product can be extracted from the entire reactionbroth with organic solvents. Product of Formula VIII is recovered bysolvent extraction. For maximum recovery, both the liquid phase filtrateand the biomass filter or centrifuge cake are treated with extractionsolvent, but usually ≧95% of the product is associated with the biomass.Typically, hydrocarbon, ester, chlorinated hydrocarbon, and ketonesolvents may be used for extraction. A preferred solvent is ethylacetate. Other typically suitable solvents include toluene and methylisobutyl ketone. For extraction from the liquid phase, it may beconvenient to use a volume of solvent approximately equal to the volumeof reaction solution which it contacts. To recover product the from thebiomass, the latter is suspended in the solvent, preferably in largeexcess relative to the initial charge of substrate, e.g., 50 to 100 ml.solvent per gram of initial canrenone charge, and the resultingsuspension preferably refluxed for a period of about 20 minutes toseveral hours to assure transfer of product to the solvent phase fromrecesses and pores of the biomass. Thereafter, the biomass is removed byfiltration or centrifugation, and the filter cake preferably washed withboth fresh solvent and deionized water. Aqueous and solvent washes arethen combined and the phases allowed to separate. Formula VIII productis recovered by crystallization from the solution. To maximize yield,the mycelium is contacted twice with fresh solvent. After settling toallow complete separation of the aqueous phase, product is recoveredfrom the solvent phase. Most preferably, the solvent is removed undervacuum until crystallization begins, then the concentrated extract iscooled to a temperature of about 0° to about 20° C., preferably about10° to about 15° C. for a time sufficient for crystal precipitation andgrowth, typically about 8 to about 12 hours.

The processes of FIG. 2, and especially that of FIG. 3, are particularlypreferred. These processes operate at low viscosity, and are amenable toclose control of process parameters such as pH, temperature anddissolved oxygen. Moreover, sterile conditions are readily preservedwithout resort to antibiotics.

The bioconversion process is exothermic, so that heat should be removed,using a jacketed fermenter or a cooling coil within the productionfermenter. Alternatively, the reaction broth may be circulated throughan external heat exchanger. Dissolved oxygen is preferably maintained ata level of at least about 5%, preferably at least about 10%, by volume,sufficient to provide energy for the reaction and assure conversion ofthe glucose to CO₂ and H₂O, by regulating the rate of air introducedinto the reactor in response to measurement of oxygen potential in thebroth. The pH is preferably controlled at between about 4.5 and about6.5.

In each of the alternative processes for 11-hydroxylation of thesubstrate of Formula XIII, productivity is limited by mass transfer fromthe solid substrate to the aqueous phase, or the phase interface, wherereaction is understood to occur. As indicated above, productivity is notsignificantly limited by mass transfer rates so long as the particlemean particle size of the substrate is reduced to less than about 300μ,and at least 75% by weight of the particles are smaller than 240μ.However, productivity of these processes may be further enhanced incertain alternative embodiments which provide a substantial charge ofcanrenone or other Formula XIII substrate to the production fermenter inan organic solvent. According to one option, the substrate is dissolvedin a water-immiscible solvent and mixed with the aqueous growth mediuminoculum and a surfactant. Useful water-immiscible solvents include, forexample, DMF, DMSO, C₆-C₁₂ fatty acids, C₆-C₁₂ n-alkanes, vegetableoils, sorbitans, and aqueous surfactant solutions. Agitation of thischarge generates an emulsion reaction system having an extendedinterfacial area for mass transfer of substrate from the organic liquidphase to the reaction sites.

A second option is to initially dissolve the substrate in a watermiscible solvent such as acetone, methylethyl ketone, methanol, ethanol,or glycerol in a concentration substantially greater than its solubilityin water. By preparing the initial substrate solution at elevatedtemperature, solubility is increased, thereby further increasing theamount of solution form substrate introduced into the reactor andultimately enhancing the reactor payload. The warm substrate solution ischarged to the production fermentation reactor along with the relativelycool aqueous charge comprising growth medium and inoculum. When thesubstrate solution is mixed with the aqueous medium, precipitation ofthe substrate occurs. However, under conditions of substantialsupersaturation and moderately vigorous agitation, nucleation is favoredover crystal growth, and very fine particles of high surface area areformed. The high surface area promotes mass transfer between the liquidphase and the solid substrate. Moreover, the equilibrium concentrationof substrate in the aqueous liquid phase is also enhanced in thepresence of a water-miscible solvent. Accordingly, productivity ispromoted.

Although the microorganism may not necessarily tolerate a highconcentration of organic solvent in the aqueous phase, a concentrationof ethanol, e.g., in the range of about 3% to about 5% by weight, can beused to advantage.

A third option is to solubilize the substrate in an aqueous cyclodextrinsolution. Illustrative cyclodextrins includehydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratioof substrate:cyclodextrin can be about 1:0.5 to about 1:1.5, morepreferably about 1:0.8 to about 1:1. The substrate:cyclodextrin mixturecan then be added aseptically to the bioconversion reactor.

11α-Hydroxycanrenone and other products of the 11α-hydroxylation process(Formulae VIII and VIIIA) are novel compounds which may be isolated byfiltering the reaction medium and extracting the product from thebiomass collected on the filtration medium. Conventional organicsolvents, e.g., ethyl acetate, acetone, toluene, chlorinatedhydrocarbons, and methyl isobutyl ketone may be used for the extraction.The product of Formula VIII may then be recrystallized from an organicsolvent of the same type. The compounds of Formula VIII have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA.

Preferably, the compounds of Formula VIII correspond to Formula VIIIA inwhich —A—A— and —B—B— are —CH₂—CH₂—, R³ is hydrogen, lower alkyl orlower alkoxy, and R⁸ and R⁹ together constitute the 20-spiroxane ring:

Further in accordance with the process of Scheme 1, the compound ofFormula VIII is reacted under alkaline conditions with a source ofcyanide ion to produce an enamine compound of Formula VII

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above. Where thesubstrate corresponds to Formula VIIIA, the product is of Formula VIIA

wherein —A—A—, —B—B—, R³, Y¹, Y², and X are as defined in Formula XIIIA.Preferably, R³ is hydrogen.

Cyanidation of the 11α-hydroxyl substrate of Formula VIII may be carriedout by reacting it with a cyanide ion source such as a ketonecyanohydrin, most preferably acetone cyanohydrin, in the presence of abase and an alkali metal salt, most preferably LiCl. Alternatively,cyanidation can be effected without a cyanohydrin by using an alkalimetal cyanide in the presence of an acid.

In the ketone cyanohydrin process, the reaction is conducted insolution, preferably using an aprotic polar solvent such asdimethylformamide or dimethyl sulfoxide. Formation of the enaminerequires at least two moles of cyanide ion source per mole of substrate,and preferably a slight excess of the cyanide source is used. The baseis preferably a nitrogenous base such as a dialkylamine, trialkylamine,alkanolamine, pyridine or the like. However, inorganic bases such asalkali metal carbonates or alkali metal hydroxides can also be used.Preferably, the substrate of Formula VIII is initially present in aproportion of between about 20 and about 50% by weight and the base ispresent in a proportion of between 0.5 to two equivalents per equivalentof substrate. The temperature of the reaction is not critical, butproductivity is enhanced by operation at elevated temperature. Thus, forexample, where triethylamine is used as the base, the reaction isadvantageously conducted in the range of about 80° C. to about 90° C. Atsuch temperatures, the reaction proceeds to completion in about 5 toabout 20 hours. When diisopropylethyl amine is used as the base and thereaction is conducted at 105° C., the reaction is completed at 8 hours.At the end of the reaction period, the solvent is removed under vacuumand the residual oil dissolved in water and neutralized to pH 7 withdilute acid, preferably hydrochloric. The product precipitates from thissolution, and is thereafter washed with distilled water and air dried.Liberated HCN may be stripped with an inert gas and quenched in analkaline solution. The dried precipitate is taken up in chloroform orother suitable solvent, then extracted with concentrated acid, e.g., 6NHCl. The extract is neutralized to pH 7 by addition of an inorganicbase, preferably an alkali metal hydroxide, and cooled to a temperaturein the range of 0° C. The resulting precipitate is washed and dried,then recrystallized from a suitable solvent, e.g., acetone, to produce aproduct of Formula VII suitable for use in the next step of the process.

Alternatively, the reaction may be conducted in an aqueous solventsystem comprising water-miscible organic solvent such as methanol or ina biphasic system comprising water and an organic solvent such as ethylacetate. In this alternative, product may be recovered by diluting thereaction solution with water, and thereafter extracting the productusing an organic solvent such as methylene chloride or chloroform, andthen back extracting from the organic extract using concentrated mineralacid, e.g., 2N HCl. See U.S. Pat. No. 3,200,113.

According to a still further alternative, the reaction may be conductedin a water-miscible solvent such as dimethylformamide,dimethylacetamide, N-methyl, pyrolidone or dimethyl sulfoxide, afterwhich the reaction product solution is diluted with water and renderedalkaline, e.g., by addition of an alkali metal carbonate, then cooled to0° to 10° C., thereby causing the product to precipitate. Preferably,the system is quenched with an alkali metal hypohalite or other reagenteffective to prevent evolution of cyanide. After filtration and washingwith water, the precipitated product is suitable for use in the nextstep of the process.

According to a still further alternative, the enamine product of FormulaVII may be produced by reaction of a substrate of Formula VIII in thepresence of a proton source, with an excess of alkali metal cyanide,preferably NaCN, in an aqueous solvent comprising an aproticwater-miscible polar solvent such as dimethylformamide ordimethylacetamide. The proton source is preferably a mineral acid or C₁to C₅ carboxylic acid, sulfuric acid being particularly preferred.Anomalously, no discrete proton source need be added where thecyanidation reagent is commercial LiCN in DMF.

A source of cyanide ion such as an alkali metal salt is preferablycharged to the reactor in a proportion of between about 2.05 and about 5molar equivalents per equivalent of substrate. The mineral acid or otherproton source is believed to promote addition of HCN across the 4,5 and6,7 double bonds, and is preferably present in a proportion of at leastone mole equivalent per mole equivalent substrate; but the reactionsystem should remain basic by maintaining an excess of alkali metalcyanide over acid present. Reaction is preferably carried out at atemperature of at least about 75° C., typically 60° C. to 100° C., for aperiod of about 1 to about 8 hours, preferably about 1.5 to about 3hours. At the end of the reaction period, the reaction mixture iscooled, preferably to about room temperature; and the product enamine isprecipitated by acidifying the reaction mixture and mixing it with coldwater, preferably at about ice bath temperature. Acidification isbelieved to close the 17-lactone, which tends to open under the basicconditions prevailing in the cyanidation. The reaction mixture isconveniently acidified using the same acid that is present during thereaction, preferably sulfuric acid. Water is preferably added in aproportion of between about 10 and about 50 mole equivalents per mole ofproduct.

The compounds of Formula VII are novel compounds and have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula VIIcorrespond to Formula VIIA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably the compound of Formula VII is5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.

In the conversion of the compound of Formula VIII to the enamine ofFormula VII, the 7-cyano derivative of the compound of Formula VIII hasbeen observed by chromatography in the crude product. It is hypothesizedthat the 7-cyano compound is an intermediate in the conversion process.It is further hypothesized that the 7-cyano intermediate itself reactsto form a second intermediate, the 5,7-dicyano derivative of thecompound of Formula VIII, which in turn reacts to form the enester. See,e.g., R. Christiansen et al., The Reaction of Steroidal 4,6-Dien-3-OnesWith Cyanide, Steroids, Vol. 1, June 1963, which is incorporated hereinby reference. These novel compounds also have utility as chromatographicmarkers as well as being synthetic intermediates. In a preferredembodiment of this step of the overall Scheme 1 synthesis process, theseintermediates are7α-cyano-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-21-dicarboxylic acid,γ-lactone, and5β,7α-dicyano-11α,17-dihydroxy-3-oxo-17α-pregnane-21-dicarboxylic acid,γ-lactone.

In the next step of the Scheme 1 synthesis, the enamine of Formula VIIis hydrolyzed to produce a diketone compound of Formula VI

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII. Anyaqueous organic or mineral acid can be used for the hydrolysis.Hydrochloric acid is preferred. To enhance productivity, awater-miscible organic solvent, such as dimethylacetamnide or a loweralkanol, is preferably used as a cosolvent. More preferably,dimethylacetamide is the solvent. The acid should be present inproportion of at least one equivalent per equivalent of Formula VIIsubstrate. In an aqueous system, the enamine substrate VII can besubstantially converted to the diketone of Formula VI in a period ofabout 5 hours at about 80° C. Operation at elevated temperatureincreases productivity, but temperature is not critical. Suitabletemperatures are selected based on the volatility of the solvent systemand acid.

Preferably, the enamine substrate of Formula VII corresponds to FormulaVIIA

and the diketone product corresponds to Formula VIA

in each of which —A—A—, —B—B—, R³, Y¹, Y², and X are as defined inFormula XIIIA. Preferably, R³ is hydrogen.

At the end of the reaction period, the solution is cooled to betweenabout 0° to 25° C. to crystallize the product. The product crystals maybe recrystallized from a suitable solvent such as isopropanol ormethanol to produce a product of Formula VI suitable for use in the nextstep of the process; but recrystallization is usually not necessary. Theproducts of Formula VI are novel compounds which have substantial valueas intermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Preferably, the compounds of Formula VIcorrespond to Formula VIA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably, the compound of Formula VI is4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile.

In a particularly preferred embodiment of the invention, the productenamine of Formula VII is produced from the compound of Formula VIII inthe manner described above, and converted in situ to the diketone ofFormula VI. In this embodiment of the invention, a formula VIIIsubstrate is reacted with an excess of alkali metal cyanide in anaqueous solvent containing a proton source, or optionally an excess ofketone cyanohydrin in the presence of a base and LiCl, as describedhereinabove. However, instead of cooling the reaction mixture,acidifying, and adding water in proportions calculated to causeprecipitation of the enamine, substantial cooling of the reactionmixture is preferably avoided. Water and an acid, preferably a mineralacid such as sulfuric, are instead added to the mixture at the end ofthe cyanidation reaction. The proportion of acid added is sufficient toneutralize excess alkali metal cyanide, which ordinarily requiresintroduction of at least one molar equivalent acid per mole of FormulaVIII substrate, preferably between about 2 and about 5 mole equivalentsper equivalent substrate. However, the temperature is maintained at highenough, and the dilution great enough, so that substantial precipitationis avoided and hydrolysis of the enamine to the diketone is allowed toproceed in the liquid phase. Thus, the process proceeds with minimuminterruption and high productivity. Hydrolysis is preferably conductedat a temperature of at least 80° C., more preferably in the range ofabout 90° C. to about 100° C., for a period of typically about 1 toabout 10 hours, more preferably about 2 to about 5 hours. Then thereaction mixture is cooled, preferably to a temperature of between about0° C. and about 15° C., advantageously in an ice bath to about 5° C. toabout 10° C., for precipitation of the product diketone of Formula VI.The solid product may be recovered, as by filtration, and impuritiesreduced by washing with water.

In the next step of the Scheme 1 synthesis, the diketone compound ofFormula VI is reacted with a metal alkoxide to open up the ketone bridgebetween the 4 and 7 positions via cleavage of the bond between thecarbonyl group and the 4-carbon, form an α-oriented alkoxycarbonylsubstituent at the 7 position, and eliminate cyanide at the 5-carbon.The product of this reaction is a hydroxyester compound corresponding toFormula V

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII, and R¹is lower alkoxycarbonyl or hydroxycarbonyl. The metal alkoxide used inthe reaction corresponds to the formula R¹⁰OM where M is alkali metaland R¹⁰O— corresponds to the alkoxy substituent of R¹. Yields of thisreaction are most satisfactory when the metal alkoxide is potassiummethoxide or sodium methoxide, but other lower alkoxides can be used. Apotassium alkoxide is particularly preferred. Phenoxides, otheraryloxides may also be used, as well as arylsulfides. The reaction isconveniently carried out in the presence of an alcohol corresponding tothe formula R¹⁰OH where R¹⁰ is as defined above. Other conventionalsolvents may be used. Preferably, the Formula VI substrate is present ina proportion of between about 2% and about 12% by weight, morepreferably at least about 6% by weight. Preferably, R¹⁰OM is present ina proportion of between about 0.5 and about 4 moles per mole ofsubstrate, more preferably between about 1 and about 2 moles per mole ofsubstrate, and still more preferably about 1.6 mole per mole ofsubstrate. Temperature is not critical but elevated temperature enhancesproductivity. Reaction time is typically between about 4 and about 24hours, preferably about 4 to 16 hours. Conveniently, the reaction iscarried out at atmospheric reflux temperature depending on the solventused.

The time required for the reaction to reach equilibrium is affected bythe amount of alkoxide that is added to the reaction mixture and themanner in which the alkoxide is added. The alkoxide may be added in asingle portion or in multiple portions or it may be added continuously.When alkoxide is added in multiple portions, it is preferable that about1.6 equivalents of potassium methoxide be added in two steps. In thistwo-step addition, 1 equivalent of potassium methoxide is initiallyadded to the reaction mixture followed by the addition of 0.6equivalents of potassium methoxide about 90 minutes later. This two-stepaddition shortens the time to reach equilibrium relative to a singleportion addition of 1.6 equivalents of potassium methoxide.

Because the equilibrium is more favorable for the production of thehydroxyester at low concentrations of the diketone, the reaction ispreferably run at rather high dilution, e.g., as high as 40:1 forreaction with sodium methoxide. It has been found that significantlyhigher productivity can be realized by use of potassium methoxide ratherthan sodium methoxide, because a dilution in the range of about 20:1 isgenerally sufficient to minimize the extent of reverse cyanidation wherepotassium methoxide is the reagent.

In accordance with the invention, it has been further discovered thatthe reverse cyanidation reaction may be inhibited by taking appropriatechemical or physical measures to remove by-product cyanide ion from thereaction zone. Thus, in a further embodiment of the invention, thereaction of the diketone with alkali metal alkoxide may be carried outin the presence of a precipitating agent for cyanide ion such as, forexample, a salt comprising a cation which forms an insoluble cyanidecompound. Such salts may, for example, include zinc iodide, ferricsulfate, or essentially any halide, sulfate or other salt of an alkalineearth or transition metal that is more soluble than the correspondingcyanide. If zinc iodide is present in proportions in the range of aboutone equivalent per equivalent diketone substrate, it has been observedthat the productivity of the reaction is increased substantially ascompared to the process as conducted in the absence of an alkali metalhalide.

Even where a precipitating agent is used for removal of cyanide ion, itremains preferable to run at fairly high dilution, but by use of aprecipitating agent the solvent to diketone substrate molar ratio may bereduced significantly compared to reactions in the absence of suchagent. Recovery of the hydroxyester of Formula V can be carried outaccording to either the extractive or non-extractive proceduresdescribed below.

The equilibrium of the reaction also can be controlled to favor theproduction of the hydroxyester of Formula V by removing thishydroxyester from the reaction mixture after it is synthesized. Theremoval of the hydroxyester can proceed either stepwise or continuouslythrough means such as filtration. The removal of the hydroxyester can beused to control the equilibrium either alone or in combination with thechemical or physical removal of cyanide from the reaction mixture.Heating of the resulting filtrate then drives the reaction equilibriumto favor of the conversion of the remaining diketone of Formula VI tothe hydroxyester of V.

In the conversion of the diketone of Formula VI to the hydroxyester ofFormula V the 5-cyano hydroxyester has been observed in the crudeproduct in small amounts, typically less than about 5% by weight. It ishypothesized that the 5-cyano hydroxyester is an equilibriumintermediate between the diketone of Formula VI and the hydroxyester ofFormula V. It is further hypothesized that this equilibrium intermediateis formed from the diketone through methoxide attack on the 5,7-oxogroup and protonation of the enolate, and from the hydroxyester througha Michael addition of by-product cyanide ion to the 3-keto-Δ^(4,5)function of the hydroxyester.

In addition, the 5-cyano-7-acid and the 17-alkoxide of the hydroxyesterof Formula V have been observed by chromatography in the crude product.It is hypothesized that the 5-cyano hydroxyester intermediate reactswith by-product cyanide ion (present as a result of the decyanationwhich introduces the Δ^(4,5) double bond) to produce the 5-cyano-7-acid.It is hypothesized that the action of the cyanide ion dealkylates the7-ester group of the 5-cyano hydroxyester to yield the 5-cyano-7-acidand the corresponding alkylnitrile.

It is further hypothesized that transient intermediate 17-alkoxide isformed from the attack of the methoxide on the 17-spirolactone of thehydroxyester (or a preceding intermediate which subsequently convertsinto the hydroxyester). The 17-alkoxide readily converts into thehydroxyester upon treatment with an acid. Therefore, it generally is notobserved in the product matrix.

The 5-cyano hydroxyester, the 5-cyano-7-acid, and the 17-alkoxide arenovel compounds which are useful as chromatographic markers and asintermediates in the preparation of the hydroxyester. They can beisolated from the crude product of this step of the Scheme 1 synthesis.Alternatively, they can be synthesized directly for use as markers orintermediates. The 5-cyano hydroxyester can be synthesized by reacting asolution of the isolated diketone of Formula VI with a base, such as analkoxide or an amine, and isolating the resulting precipitate. Thecompound prepared preferably is 7-methyl hydrogen5β-cyano-11α,17-dihydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone.

The 5-cyano-7-carboxylic acid can be synthesized directly by reactingthe diketone of Formula VI with a weak aqueous base, such as sodiumacetate or sodium bicarbonate, and isolating the resulting precipitate.The compound prepared preferably is5-β-cyano-11-α,17-dihydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylic acid,γ-lactone.

The 17-alkoxide can be synthesized directly by reacting a solution ofthe hydroxyester of Formula V with an alkoxide to yield a mixture of the17-alkoxide and the corresponding hydroxyester. The compound preparedpreferably is dimethyl11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate, γ-lactone.

Preferably, the diketone substrate of Formula VI corresponds to FormulaVIA

and the hydroxyester product corresponds to Formula VA

in each of which —A—A—, —B—B—, R³, Y¹, Y², and X are as defined inFormula XIIIA and R¹ is as defined in Formula V. Preferably, R³ ishydrogen.

The products of Formula V are novel compounds which have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula Vcorrespond to Formula VA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably, the compound of Formula V is Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

The compound of Formula V may be isolated by filtration or by acidifyingthe reaction solution, e.g., with a mineral acid such as aqueous HCl orsulfuric acid, cooling to ambient temperature, and extracting theproduct with an organic solvent such as methylene chloride or ethylacetate. The extract is washed with an aqueous alkaline wash solution,dried and filtered, after which the solvent is removed. Alternatively,the reaction solution containing the product of Formula V may bequenched with concentrated acid. The product solution is concentrated,cooled to between about 0° to 25° C. and the product solid is isolatedby filtration.

In a preferred embodiment, methanol and HCN are removed by distillationafter the conclusion of the reaction period, with mineral acid (such ashydrochloric acid or sulfuric acid) being added before the distillationand water being added after the distillation. The mineral acid can beadded in a single step, in multiple steps or continuously. In apreferred embodiment, mineral acid is continuously added over a periodof about 10 to about 40 minutes, more preferably about 15 to about 30minutes. Likewise, water can be added to the still bottoms in a singlestep, in multiple steps or continuously. In a preferred embodiment, theconcentrated reaction mixture is cooled from reflux temperature prior toaddition of water. Preferably, the mixture is cooled to a temperaturebetween about 50° C. to about 70° C., preferably between about 60° C. toabout 70° C., and more preferably about 65° C., prior to addition of thewater. Water is then added, preferably continuously over a period ofabout 15 minutes to about 3 hours, and more preferably over about 60minutes to about 90 minutes, while maintaining the temperatureapproximately constant. Product of Formula V begins to crystallize fromthe still bottoms as the water addition proceeds. After the water hasbeen added to the mixture, the diluted reaction mixture is maintained atabout the same temperature for about 1 hour and then cooled to about 15°C. over an additional period of about 4 to about 5 hours. The mixture ismaintained at about 15° C. for a period of about 1 to 2 hours. A longerholding period at 15° C. increases the yield of the cyanoester in themixture. This mode of recovery provides a high quality crystallineproduct without extraction operations.

According to another preferred mode of recovery of the product ofFormula V, methanol and HCN are removed by distillation after theconclusion of the reaction period, with water and acid being addedbefore or during the distillation. Addition of water before thedistillation simplifies operations, but progressive addition during thedistillation allows the volume in the still to be maintainedsubstantially constant. Product of Formula V crystallizes from the stillbottoms as the distillation proceeds. This mode of recovery provides ahigh quality crystalline product without extraction operations.

In accordance with yet a further alternative, the reaction solutioncontaining the product of Formula V may be quenched with mineral acid,e.g., 4N HCl, after which the solvent is removed by distillation.Removal of the solvent is also effective for removing residual HCN fromthe reaction product. It has been found that multiple solventextractions for purification of the compound of Formula V are notnecessary where the compound of Formula V serves as an intermediate in aprocess for the preparation of epoxymexrenone, as described herein. Infact, such extractions can often be entirely eliminated. Where solventextraction is used for product purification, it is desirable tosupplement the solvent washes with brine and caustic washes. But wherethe solvent extractions are eliminated, the brine and caustic washes aretoo. Eliminating the extractions and washes significantly enhances theproductivity of the process, without sacrificing yield or productquality, and also eliminates the need for drying of the washed solutionwith a dessicant such as sodium sulfate.

The crude 11α-hydroxy-7α-alkoxycarbonyl product is taken up again in thesolvent for the next reaction step of the process, which is theconversion of the 11-hydroxy group to a leaving group at the 11 positionthereby producing a compound of Formula IV:

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula XIII, R¹ isas defined in Formula V, and R² is lower arylsulfonyloxy,alkylsulfonyloxy, acyloxy or halide. Preferably, the 11α-hydroxy isesterified by reaction with a lower alkylsulfonyl halide, an acyl halideor an acid anhydride which is added to the solution containing theintermediate product of Formula V. Lower acid anhydrides such as aceticanhydride and trihalogenated acid anhydrides such as trifluoroaceticanhydride can be used to prepare suitable acyloxy leaving groups. Loweralkylsulfonyl halides, and especially methanesulfonyl chloride, however,are preferred. Alternatively, the 11-α hydroxy group could be convertedto a halide by reaction of a suitable reagent such as thionyl bromide,thionyl chloride, sulfuryl chloride or oxalyl chloride. Other reagentsfor forming 11α-sulfonic acid esters include tosyl chloride,benzenesulfonyl chloride and trifluoromethanesulfonic anhydride. Thereaction is conducted in a solvent containing a hydrogen halidescavenger such as triethylamine or pyridine. Inorganic bases such aspotassium carbonate or sodium carbonate can also be used. The initialconcentration of the hydroxyester of Formula V is preferably betweenabout 5% and about 50% by weight. The esterification reagent ispreferably present in slight excess. Methylene chloride is aparticularly suitable solvent for the reaction, but other solvents suchas dichloroethane, pyridine, chloroform, methyl ethyl ketone,dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers,acetonitrile, toluene, and tetrahydrofuran can also be employed. Thereaction temperature is governed primarily by the volatility of thesolvent. In methylene chloride, the reaction temperature is preferablyin the range of between about −10° C. and about 10° C.

Preferably, the hydroxyester substrate of Formula V corresponds toFormula VA

and the product corresponds to Formula IVA

in each of which —A—A—, —B—B—, R³, Y¹, Y², and X are as defined inFormula XIIIA, R¹ is lower alkoxycarbonyl or hydroxycarbonyl, and R² isas defined in Formula IV. Preferably, R³ is hydrogen.

The products of Formula IV are novel compounds which have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula IVAcorrespond to Formula VA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably, the compound of Formula IV is Methyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone. Where an acyloxy leaving group is desired, the compound ofFormula IV is preferably 7-methyl hydrogen17-hydroxy-3-oxo-11α-(2,2,2-trifluoro-1-oxoethoxy)-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone; or 7-methyl11α-(acetyloxy)-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone.

If desired, the compound of Formula IV may be isolated by removal of thesolvent. Preferably, the reaction solution is first washed with anaqueous alkaline wash solution, e.g., 0.5-2N NaOH, followed by an acidwash, e.g., 0.5-2N HCl. After removal of the reaction solvent, theproduct is recrystallized, e.g., by taking the product up in methylenechloride and then adding another solvent such as ethyl ether whichlowers the solubility of the product of Formula IV, causing it toprecipitate in crystalline form.

In the recovery of the product of Formula IV, or in preparation of thereaction solution for conversion of the Formula IV intermediate to theintermediate of Formula II as is further described hereinbelow, allextractions and/or washing steps may be dispensed with if the solutionis instead treated with ion exchange resins for removal of acidic andbasic impurities. The solution is treated first with an anion exchangeresin, then with a cation exchange resin. Alternatively, the reactionsolution may first be treated with inorganic adsorbents such as basicalumina or basic silica, followed by a dilute acid wash. Basic silica orbasic alumina may typically be mixed with the reaction solution in aproportion of between about 5 and about 50 g per kg of product,preferably between about 15 and about 20 g per kg product. Whether ionexchange resins or inorganic adsorbents are used, the treatment can becarried out by simply slurrying the resin or inorganic adsorbent withthe reaction solution under agitation at ambient temperature, thenremoving the resin or inorganic adsorbent by filtration.

In an alternative and preferred embodiment of the invention, the productcompound of Formula IV is recovered in crude form as a concentratedsolution by removal of a portion of the solvent. This concentratedsolution is used directly in the following step of the process, which isremoval of the 11α-leaving group from the compound of Formula IV,thereby producing an enester of Formula II:

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII, and R¹is as defined in Formula V. For purposes of this reaction, the R²substituent of the compound of Formula IV may be any leaving group theabstraction of which is effective for generating a double bond betweenthe 9- and 11-carbons. Preferably, the leaving group is a loweralkylsulfonyloxy or acyloxy substituent which is removed by reactionwith an acid and an alkali metal salt. Mineral acids can be used, butlower alkanoic acids are preferred. Advantageously, the reagent for thereaction further includes an alkali metal salt of the alkanoic acidutilized. It is particularly preferred that the leaving group comprisemesyloxy and the reagent for the reaction comprise formic acid or aceticacid and an alkali metal salt of one of these acids or another loweralkanoic acid. Where the leaving group is mesyloxy and the removalreagent is either acetic acid and sodium acetate or formic acid andpotassium formate, a relatively high ratio of 9,11-olefin to11,12-olefin is observed. If free water is present during removal of theleaving group, impurities tend to be formed, particularly a 7,9-lactone

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula XIII, whichis difficult to remove from the final product. Hence, acetic anhydrideor other drying agent is used to remove the water present in formicacid. The free water content of the reaction mixture before reactionshould be maintained at a level below about 0.5%, preferably below about0.1% by weight, as measured by Karl Fischer analysis for water, based ontotal reaction solution. Although it is preferred that the reactionmixture be kept as dry as practicable, satisfactory results have beenrealized with 0.3% by weight water. Preferably, the reaction chargemixture contains between about 4% and about 50% by weight of thesubstrate of Formula IV in the alkanoic acid. Between about 4% and about20% by weight of the alkali metal salt of the acid is preferablyincluded. Where acetic anhydride is used as the drying agent, it ispreferably present in a proportion of between about 0.05 moles and about0.2 moles per mole of alkanoic acid.

It has been found that proportions of by-product 7,9-lactone and11,12-olefin in the reaction mixture is relatively low where theelimination reagent comprises a combination of trifluoroacetic acid,trifluoroacetic anhydride and potassium acetate as the reagent forelimination of the leaving group and formation of the enester(9,11-olefin). Trifluoroacetic anhydride serves as the drying agent, andshould be present in a proportion of at least about 3% by weight, morepreferably at least about 15% by weight, most preferably about 20% byweight, based on the trifluoroacetic acid eliminating reagent.

In addition to the 7,9-lactone, other impurities and by-products whichare useful as synthetic intermediates and chromatographic markers havebeen observed in this step of the Scheme 1 synthesis. The novel4,9,13-triene of the enester of Formula II (for example, 7-methylhydrogen17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7α,21-dicarboxylate) hasbeen isolated chromatographically from the product solution. The amountof this compound produced appears to increase with an increase inreaction time for this step of the synthesis. It is hypothesized thatthe compound is formed when the lactone is protonated and the resultingC17 carbonium ion facilitates the migration of the angular methyl groupfrom the C13 position. Deprotonation of this intermediate yields the4,9,13-triene.

The novel 5-cyano-Δ^(11,12) of the enester of Formula II (for example,7-methyl hydrogen5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate,γ-lactone) and the novel 5-cyano of the enester of Formula II (forexample, 7-methyl hydrogen5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate,γ-lactone) also have been isolated chromatographically from the crudeproduct. It is hypothesized that these compounds are formed viadehydration of the residual 5-cyano-7-acid and 5-cyano hydroxyester,respectively, which are present in the crude product solution as aresult of the third step of the Scheme 1 synthesis.

The novel C17 epimer of the enester of Formula II (for example, 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate,γ-lactone) also has been isolated chromatographically from the crudeproduct. It is hypothesized that the acidic conditions of theelimination reaction can result in racemization of the C17 chiral centerto yield the 17-epimer of the enester. The 17-epimer can be synthesizeddirectly by reacting a compound of Formula IV with a solution ofpotassium formate, formic acid and acetic anhydride and isolating the17-epimer.

Although not observed as an impurity in the crude product solution, the11-ketone of the hydroxyester of formula V can be prepared by oxidizingthe 11-hydroxy of the corresponding hydroxyester with a suitableoxidizing agent such as a Jones Reagent. The 11-ketone preparedpreferably is 7-methyl hydrogen17-hydroxy-3,11-dioxo-17α-pregna-4-ene-7α,21-dicarboxylate, γ-lactone.

Alternatively, the 11α-leaving groups from the compound of Formula IV,may be eliminated to produce an enester of Formula II by heating asolution of Formula IV in an organic solvent such as DMSO, DMF or DMA.

Further in accordance with the invention, the compound of Formula IV isreacted initially with an alkenyl alkanoate such as isopropenyl acetatein the presence of an acid such as toluene sulfonic acid or an anhydrousmineral acid such as sulfuric acid to form the 3-enol ester:

of the compound of Formula IV. Alternatively, the 3-enol ester can beformed by treatment of the compound of Formula IV with an acid anhydrideand base such as acetic acid and sodium acetate. Further alternativesinclude treatment of the compound of Formula IV with ketene in thepresence of an acid to produce the compound of Formula IV(Z). Theintermediate of Formula IV(Z) is thereafter reacted with an alkali metalformate or acetate in the presence of formic or acetic acid to producethe Δ^(9,11) enol acetate of Formula IV(Y)

which can then be converted to the enester of Formula II in an organicsolvent, preferably an alcohol such as methanol, by either thermaldecomposition of the enol acetate or reaction thereof with an alkalimetal alkoxide. The elimination reaction is highly selective to theenester of Formula II in preference to the 11,12-olefin and 7,9-lactone,and this selectivity is preserved through conversion of the enol acetateto the enone.

Preferably, the substrate of Formula IV corresponds to Formula IVA

and the enester product corresponds to Formula IIA

in each of which —A—A—, —B—B—, R₃, Y¹, Y², and X are as defined inFormula XIIIA, and R¹ is as defined in Formula V. Preferably, R₃ ishydrogen.

If desired, the compound of Formula II may be isolated by removing thesolvent, taking up the solid product in cold water, and extracting withan organic solvent, such as ethyl acetate. After appropriate washing anddrying steps, the product is recovered by removing the extractionsolvent. The enester is then dissolved in a solvent appropriate for theconversion to the product of Formula I. Alternatively, the enester canbe isolated by adding water to the concentrated product solution andfiltering the solid product, thereby preferentially removing the7,9-lactone. Conversion of the substrate of Formula II to the product ofFormula IA may be conducted in the manner described in U.S. Pat. No.4,559,332 which is expressly incorporated herein by reference, or morepreferably by the novel reaction using a haloacetamide promoter asdescribed below.

In another embodiment of the invention, the hydroxyester of Formula Vmay be converted to the enester of Formula II without isolation of theintermediate compound of Formula IV. In this method, the hydroxyester istaken up in an organic solvent, such as methylene chloride; and eitheran acylating agent, e.g., methanesulfonyl chloride, or halogenatingreagent, e.g., sulfuryl chloride, is added to the solution. The mixtureis agitated and, where halogenation is involved, an HCl scavenger suchas imidazole is added. This reaction is highly exothermic, and shouldtherefore be conducted at a controlled rate with full cooling. After thebase addition, the resulting mixture is warmed to moderate temperature,e.g., about 0° C. to room temperature or slightly above, and reacted fora period of typically about 1 to about 4 hours. After reaction iscomplete, the solvent is stripped, preferably under high vacuum (e.g.,about 24″ to about 28″ Hg) conditions at about −10° to about +15° C.,more preferably about 0° to about 5° C., to concentrate the solution andremove excess base The substrate is then redissolved in an organicsolvent, preferably a halogenated solvent such as methylene chloride forconversion to the enester.

The leaving group elimination reagent is preferably prepared by mixingan organic acid, an organic acid salt and a drying agent, preferablyformic acid, alkali metal formate and acetic anhydride, respectively, ina dry reactor. Addition of acetic anhydride is exothermic and results inrelease of CO, so the addition rate must be controlled accordingly. Topromote the removal of water, the temperature of this reaction ispreferably maintained in the range of about 60° to about 90° C., mostpreferably about 65° to about 75° C. This reagent is then added to theproduct solution of the compound of Formula IV to effect the eliminationreaction. After about 4 to about 8 hours, the reaction mixture ispreferably heated to a temperature of at least about 85° C., butpreferably not above about 95° C. until all volatile distillate has beenremoved, and then for an additional period to complete the reaction,typically about 1 to about 4 hours. The reaction mixture is cooled, andafter recovery by standard extraction techniques, the enester may berecovered as desired by evaporating the solvent.

It has further been found that the enester of Formula II may berecovered from the reaction solution by an alternative procedure whichavoids the need for extraction steps following the elimination reaction,thereby providing savings in cost, improvement in yield and/orimprovement in productivity. In this process, the enester product isprecipitated by dilution of the reaction mixture with water afterremoval of formic acid. The product is then isolated by filtration. Noextractions are required.

According to a further alternative for conversion of the hydroxyester ofFormula V to the enester of Formula II without isolation of the compoundof Formula IV, the 11α-hydroxy group of the Formula V hydroxyester isreplaced by halogen, and the Formula II enester is then formed in situby thermal dehydrohalogenation. Replacement of the hydroxy group byhalogen is effected by reaction with sulfuryl halide, preferablysulfuryl chloride, in the cold in the presence of a hydrogen halidescavenger such as imidazole. The hydroxyester is dissolved in a solventsuch as tetrahydrofuran and cooled to about 0° C. to about −70° C. Thesulfuryl halide is added and the reaction mixture is warmed to moderatetemperature, e.g., room temperature, for a time sufficient to completethe elimination reaction, typically about 1 to about 4 hours. Theprocess of this embodiment not only combines two steps into one, buteliminates the use of: a halogenated reaction solvent; an acid (such asacetic acid); and a drying reagent (such as acetic anhydride or sodiumsulfate). Moreover, the reaction does not require refluxing conditions,and avoids the generation of by-product CO which results when aceticacid is used as a drying reagent.

In accordance with a particularly preferred embodiment of the invention,the diketone compound of Formula VI can be converted to epoxymexrenoneor other compound of Formula I without isolating any intermediate inpurified form. In accordance with this preferred process, the reactionsolution containing the hydroxyester is quenched with a strong acidsolution, cooled to ambient temperature and then extracted with anappropriate extraction solvent. Advantageously, an aqueous solution ofinorganic salt, e.g., about 10% by weight saline solution, is added tothe reaction mixture prior to the extraction. The extract is washed anddried by azeotropic distillation for removal of the methanol solventremaining from the ketone cleavage reaction.

The resulting concentrated solution containing between about 5% andabout 50% by weight compound of Formula V is then contacted in the coldwith an acylating or alkylsulfonylating reagent to form the sulfonicester or dicarboxylic acid ester. After the alkylsulfonation orcarboxylation reaction is complete, the reaction solution is passed overan acidic and then a basic exchange resin column for the removal ofbasic and acidic impurities. After each pass, the column is washed withan appropriate solvent, e.g., methylene chloride, for the recovery ofresidual sulfonic or dicarboxylic ester therefrom. The combined eluateand wash fractions are combined and reduced, preferably under vacuum, toproduce a concentrated solution containing the sulfonic ester ordicarboxylic ester of Formula IV. This concentrated solution is thencontacted with a dry reagent comprising an agent effect for removal ofthe 11α-ester leaving group and abstraction of hydrogen to form a 9,11double bond. Preferably, the reagent for removal of the leaving groupcomprises the formic acid/alkali metal formate/acetic anhydride dryreagent solution described above. After reaction is complete, thereaction mixture is cooled and formic acid and/or other volatilecomponents are removed under vacuum. The residue is cooled to ambienttemperature, subjected to appropriate washing steps, and then dried togive a concentrated solution containing the enester of Formula II. Thisenester may then be converted to epoxymexrenone or other compound ofFormula I using the method described herein, or in U.S. Pat. No.4,559,332.

In an especially preferred embodiment of the invention, the solvent isremoved from the reaction solution under vacuum, and the product ofFormula IV is partitioned between water and an appropriate organicsolvent, e.g., ethyl acetate. The aqueous layer is then back extractedwith the organic solvent, and the back extract washed with an alkalinesolution, preferably a solution of an alkali metal hydroxide containingan alkali metal halide. The organic phase is concentrated, preferablyunder vacuum, to yield the enester product of Formula II. The product ofFormula II may then be taken up in an organic solvent, e.g., methylenechloride, and further reacted in the manner described in the '332 patentto produce the product of Formula I.

Where trihaloacetonitrile is used in the epoxidation reaction, it hasbeen found that the selection of solvent is important, with halogenatedsolvents being highly preferred, and methylene chloride being especiallypreferred. Solvents such as dichloroethane and chlorobenzene givereasonably satisfactory yields, but yields are generally better in amethylene chloride reaction medium. Solvents such as acetonitrile andethyl acetate generally give poor yields, while reaction in solventssuch as methanol or water/tetrahydrofuran give little of the desiredproduct.

Further in accordance with the present invention, it has been discoveredthat numerous improvements in the synthesis of epoxymexrenone can berealized by use of a trihaloacetamide rather than a trihaloacetonitrileas a peroxide activator for the epoxidation reaction. In accordance witha particularly preferred process, the epoxidation is carried out byreaction of the substrate of Formula IIA with hydrogen peroxide in thepresence of trichloroacetamide and an appropriate buffer. Preferably,the reaction is conducted in a pH in the range of about 3 to about 7,most preferably between about 5 and about 7. However, despite theseconsiderations, successful reaction has been realized outside thepreferred pH ranges.

Especially favorable results are obtained with a buffer comprisingdipotassium hydrogen phosphate, and/or with a buffer comprising acombination of dipotassium hydrogenphosphate and potassium dihydrogenphosphate in relative proportions of between about 1:4 and about 2:1,most preferably in the range of about 2:3. Borate buffers can also beused, but generally give slower conversions than dipotassium phosphateor K₂HPO₄/KH₂PO₄ mixtures. Whatever the makeup of the buffer, it shouldprovide a pH in the range indicated above. Aside from the overallcomposition of the buffer or the precise pH it may impart, it has beenobserved that the reaction proceeds much more effectively if at least aportion of the buffer is comprised of dibasic hydrogenphosphate ion. Itis believed that this ion may participate essentially as a homogeneouscatalyst in the formation of an adduct or complex comprising thepromoter and hydroperoxide ion, the generation of which may in turn beessential to the overall epoxidation reaction mechanism. Thus, thequantitative requirement for dibasic hydrogenphosphate (preferably fromK₂HPO₄) may be only a small catalytic concentration. Generally, it ispreferred that K₂HPO₄ be present in a proportion of at least about 0.1equivalents, e.g., between about 0.1 and about 0.3 equivalents, perequivalent substrate.

The reaction is carried out in a suitable solvent, preferably methylenechloride, but alternatively other halogenated solvents such aschlorobenzene or dichloroethane can be used. Toluene and mixtures oftoluene and acetonitrile have also been found satisfactory. Withoutcommitting to a particular theory, it is posited that the reactionproceeds most effectively in a two phase system in which a hydroperoxideintermediate is formed and distributes to the organic phase of low watercontent, and reacts with the substrate in the organic phase. Thus thepreferred solvents are those in which water solubility is low. Effectiverecovery from toluene is promoted by inclusion of another solvent suchas acetonitrile.

In the conversion of substrates of Formula II to products of Formula I,toluene provides a process advantage since the substrates are freelysoluble in toluene and the products are not. Thus, the productprecipitates during the reaction when conversions reach the 40-50%range, producing a three phase mixture from which the product can beconveniently separated by filtration. Methanol, ethyl acetate,acetonitrile alone, THF and THF/water have not proved to be as effectiveas the halogenated solvents or toluene in carrying out the conversion ofthis step of the process.

While trichloroacetamide is a highly preferred reagent, othertrihaloacetamides such as trifluoroacetamide and chlorodifluoroacetamidecan also be used. Trihalomethylbenzamide, and other compounds having anarylene, alkenyl or alkynyl moiety (or other group which allows thetransfer of the electron withdrawing effect of the electron withdrawinggroup to the amide carbonyl) between the electron withdrawingtrihalomethyl group and the carbonyl of the amide, may also be useful.Heptafluorobutyramides may also be used, but with less favorableresults. Generically, the peroxide activator may correspond to theformula:

R^(o)C(O)NH₂

where R^(o) is a group having an electron withdrawing strength (asmeasured by sigma constant) at least as high as that of themonochloromethyl group. The electron withdrawing group preferably isattached directly to the amide carbonyl for maximum effectiveness. Moreparticularly, the peroxide activator may correspond to the formula:

where R^(p) is a group which allows the transfer of the electronwithdrawing effect of an electron withdrawing group to the amidecarbonyl, and preferably is selected from among arylene, alkenyl,alkynyl and —(CX⁴X⁵)_(n)— moieties; X¹, X², X³, X⁴ and X⁵ areindependently selected from among halo, hydrogen, alkyl, haloalkyl andcyano and cyanoalkyl; and n is 0, 1 or 2; provided that when n is 0,then at least one of X¹, X² and X³ is halo; and when R^(p) is—(CX⁴X⁵)_(n)— and n is 1 or 2, then at least one of X⁴ and X⁵ is halo.Where any of X¹, X², X³, X⁴ or X⁵ is not halo, it is preferablyhaloalkyl, most preferably perhaloalkyl. Particularly preferredactivators include those in which n is 0 and at least two of X¹, X² andX³ are halo; or those in which R^(p) is —(CX⁴X⁵)_(n)—, n is 1 or 2, atleast one of X⁴ and X⁵ is halo, the other of X⁴ and X⁵ is halo orperhaloalkyl, and X¹, X² and X³ are halo or perhaloalkyl. Each of X¹, X²X³, X⁴ and X⁵ is preferably Cl or F, most preferably Cl, though mixedhalides may also be suitable, as may perchloralkyl or perbromoalkyl andcombinations thereof, provided that the carbon directly attached to theamide carbonyl is substituted with at least one halo group.

Preferably, the peroxide activator is present in a proportion of atleast about 1 equivalent, more preferably between about 1.5 and about 2equivalents, per equivalent of substrate initially present. Hydrogenperoxide should be charged to the reaction in at least modest excess, oradded progressively as the epoxidation reaction proceeds. Although thereaction consumes only one to two equivalents of hydrogen peroxide permole of substrate, hydrogen peroxide is preferably charged insubstantial excess relative to substrate and activator initiallypresent. Without limiting the invention to a particular theory, it isbelieved that the reaction mechanism involves formation of an adduct ofthe activator and the peroxide anion, that the formation of thisreaction is reversible with the equilibrium favoring the reversereaction, and that a substantial initial excess of hydrogen peroxide istherefore necessary in order to drive the reaction in the forwarddirection. Temperature of the reaction is not narrowly critical, and maybe effectively carried out within the range of about 0° to about 100° C.The optimum temperature depends on the selection of solvent. Generally,the preferred temperature is between about 20° C. and about 30° C., butin certain solvents, e.g., toluene the reaction may be advantageouslyconducted in the range of about 60° to about 70° C. At about 25° C.,reaction typically requires less than about 10 hours, typically about 3to about 6 hours. If needed, additional activator and hydrogen peroxidemay be added at the end of the reaction cycle to achieve completeconversion of the substrate.

At the end of the reaction cycle, the aqueous phase is removed, theorganic reaction solution is preferably washed for removal of watersoluble impurities, after which the product may be recovered by removalof the solvent. Before removal of solvent, the reaction solution shouldbe washed with at least a mild to moderately alkaline wash, e.g., sodiumcarbonate. Preferably, the reaction mixture is washed successively with:a mild reducing solution such as a weak (e.g. about 3% by weight)solution of sodium sulfite in water; an alkaline solution, e.g., NaOH orKOH (preferably about 0.5N); an acid solution such as HCl (preferablyabout 1N); and a final neutral wash comprising water or brine,preferably saturated brine to minimize product losses. Prior to removalof the reaction solvent, another solvent such as an organic solvent,preferably ethanol may be advantageously added, so that the product maybe recovered by crystallization after distillation for removal of themore volatile reaction solvent.

It should be understood that the novel epoxidation method utilizingtrichloroacetamide or other novel peroxide activator has applicationwell beyond the various schemes for the preparation of epoxymexrenone,and in fact may be used for the formation of epoxides across olefinicdouble bonds in a wide variety of substrates subject to reaction in theliquid phase. The reaction is particularly effective in unsaturatedcompounds in which the olefins are tetrasubstituted and trisubstituted,i.e., R^(a)R^(b)C═CR^(c)R^(d) and R^(a)R^(b)C═CR^(c)H where R^(a) toR^(d) represent substituents other than hydrogen. The reaction proceedsmost rapidly and completely where the substrate is a cyclic compoundwith a trisubstituted double bond, or either a cyclic or acycliccompound with a tetrasubstituted double bond. Exemplary substrates forthe epoxidation reaction include Δ^(9,11)-canrenone, and the followingsubstrates:

Because the reaction proceeds more rapidly and completely withtrisubstituted and tetrasubstituted double bonds, it is especiallyeffective for selective epoxidation across such double bonds incompounds that may include other double bonds where the olefinic carbonsare monosubstituted, or even disubstituted.

Other non-limiting examples illustrating the generic epoxidationreaction include the following epoxidation reactions:

It should be further understood that the reaction may he used toadvantage in the epoxidation of monosubstituted or even disubstituteddouble bonds, such as the 11,12-olefin in various steroid substrates.However, because it preferentially epoxidizes the more highlysubstituted double bonds, e.g., the 9,11-olefin, with high selectivity,the process of this invention is especially effective for achieving highyields and productivity in the epoxidation steps of the various reactionschemes described elsewhere herein.

The improved process has been shown to be a particularly advantageousapplication to the preparation of:

the epoxidation of:

the preparation of:

the epoxidation of:

Multiple advantages have been demonstrated for the process of theinvention in which trichloroacetamide is used in place oftrichloroacetonitrile as the oxygen transfer reagent for the epoxidationreaction. The trichloroacetamide reagent system has a low affinity forelectronically deficient olefins such as α,β-unsaturated ketones. Thisallows for selective epoxidation of a non-conjugated olefin insubstrates containing both types of double bonds. Additionally, incomplex substrates such as steroids, disubstituted and trisubstitutedolefins can be differentiated by reaction. Thus, good selectivity isobserved in the epoxidation of the isomeric Δ-9,11 and Δ-11,12compounds. In this case, the 9,11 epoxide is formed with minimalreaction of the isomer containing the Δ-11,12 double bond. Accordingly,reaction yield, product profile and final purity are substantiallyenhanced in comparison to reactions in which a trihaloacetonitrile isused. It has further been discovered that the substantial excess oxygengeneration observed with the use of trihaloacetonitrile is minimizedwith trichloroacetamide, imparting improved safety to the epoxidationprocess. Further in contrast to the trichloroacetonitrile promotedreaction, the trichloroacetamide reaction exhibits minimum exothermiceffects, thus facilitating control of the thermal profile of thereaction. Agitation effects are observed to be minimal and reactorperformance more consistent, a further advantage over thetrichloroacetonitrile process. The reaction is more amenable to scaleupthan the trichloroacetonitrile promoted reaction. Product isolation andpurification is simple. There is no observable Bayer-Villager oxidationof carbonyl function (peroxide promoted conversion of ketone to ester)as experienced when using m-chloroperoxybenzoic acid or other peracids.The reagent is inexpensive, readily available, and easily handled.

In addition, the following compounds have been observed bychromatography in the crude product from the step of the Scheme 1synthesis in which the enester of Formula II is converted to thecompound of Formula I:

(1) the novel 11α,12α epoxide of the enester of formula II, for example,7-methyl hydrogen11α,12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone;

(2) the novel 4,5:9,11-diepoxide of the enester of formula II, forexample 7-methyl hydrogen4α,5α:9α,11α-diepoxy-17-hydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone;

(3) the novel 12-ketone of the enester of formula II, for example7-methyl hydrogen17-hydroxy-3,12-dioxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone;

(4) the novel 9,11-dihydroxy of the enester of formula II, for example7-methyl hydrogen9α,11β,17-trihydroxy-3-oxo-17α-pregna-4-ene-7α,21-dicarboxylate,γ-lactone;

(5) the novel 12-hydroxy analog of the enester of formula II, forexample 7-methyl hydrogen12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone; and

(6) the novel 7-acid of the compound of Formula I, for example9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,γ-lactone.

These compounds have utility as synthetic intermediates and/orchromatographic markers in the preparation of the compound of Formula I,particularly epoxymexrenone.

The 11α,12α-epoxide of the enester of formula II is hypothesized to formvia an impurity produced during the previous step in which a compound offormula IV is converted to the enester of formula II. This impurity waschromatographically isolated and is the Δ^(11,12) enester. It typicallyis produced with the Δ^(9,11) enester in a ratio of about 90:10(Δ^(9,11) enester:Δ^(11,12) enester), although this ratio can vary.Oxidation of the Δ^(11,12) enester during the conversion of the enesterof formula II to the compound of Formula I yields the 11α,12α-epoxide.

The 4,5:9,11-diepoxide of the enester of Formula I waschromatographically isolated. It is hypothesized to result fromover-epoxidation of the enester. It typically is observed in the crudeproduct at levels of about 5% by weight or less, although this amountcan vary.

The 12-ketone of the enester of Formula II was chromatographicallyisolated. It is hypothesized to result from allylic oxidation of theenester. It typically is observed in the crude product at levels ofabout 5% by weight or less, although this amount can vary. The level of12-ketone detected in the crude product when trichloroacetonitrile wasused as the hydrogen peroxide activator was higher than the leveldetected when trichloroacetamide was used as the activator.

The 9,11-dihydroxy of the enester of Formula II was chromatographicallyisolated. It typically is observed in the crude product at levels ofabout 5% by weight or less, although this amount can vary. It ishypothesized to result from hydrolysis of the epoxide of Formula I.

The 12-hydroxy of the enester of formula II was chromatographicallyisolated. It typically is observed in the crude product at levels ofabout 5% by weight or less, although this amount can vary. It ishypothesized to result from hydrolysis of the 11,12 epoxide withsubsequent elimination of the 11β-hydroxy.

In addition, the compounds of Formula I prepared in accordance with thisdisclosure can be further modified to provide a metabolite, derivative,prodrug or the like with improved properties (such as improvedsolubility and absorption) which facilitate the administration and/orefficacy of epoxymexrenone. The 6-hydroxy of the compound of Formula I(for example, 7-methyl hydrogen6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone) is a novel compound which has been identified as a possiblemetabolite in the rat. The 6-hydroxy metabolite can be prepared from thecorresponding ethyl enol ether (for example, 7-methyl hydrogen9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone). The ethyl enol ether of of the compound of Formula I can beprepared in accordance with the procedure set forth in R. M. Weier andL. M. Hofmann (J. Med Chem 1977, 1304) which is incorporated byreference herein. The ethyl enol ether is then reacted withm-chloroperbenzoic acid to yield the corresponding 6-hydroxy of thecompound of Formula I.

It is further hypothesized that the monocarboxylic salts ofepoxymexrenone, particularly the potassium and sodium salts, aresuitable alternatives to facilitate administration of a compound ofFormula I to an individual for whom administration of an aldosteroneantagonist is indicated. Under mild basic conditions it is possible toselectively open the spirolactone of the compounds of Formula I withouthydrolyzing the C7 ester substituent to give the corresponding17β-hydroxy-17α-(3-propionic acid) analog. These open chain analogs aremore polar than their lactone counterparts and have shorter retentiontimes when analyzed by reverse phase HPLC. Acidic conditions generallycause the regeneration of the lactone ring.

Under more forcing conditions, the spirolactone is opened and the C7ester is hydrolyzed to give the corresponding by-products,17β-hydroxy-17α-(3-propionic acid)-7-acid analogs of the compounds ofFormula I. These dicarboxylic acids have shorter retention times thanthe monocarboxylic acids when analyzed by reverse phase HPLC. Acidicconditions (e.g., treatment with a dilute acid such as 0.1-4 Mhydrochloric acid) generally cause the regeneration of the lactone ringof the dicarboxylic acid.

The novel epoxidation method of the invention is highly useful as theconcluding step of the synthesis of Scheme 1. In a particularlypreferred embodiment, the overall process of Scheme 1 proceeds asfollows:

The second of the novel reaction schemes (Scheme 2) of this inventionstarts with canrenone or other substrate corresponding to Formula XIII

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII. In thefirst step of this process, the substrate of Formula XIII is convertedto a product of Formula XII

using a cyanidation reaction scheme substantially the same as thatdescribed above for conversion of the substrate of Formula VIII to theintermediate of Formula VII. Preferably, the substrate of Formula XIIIcorresponds to Formula XIIIA

and the enamine product corresponds to Formula XIIA

in each of which —A—A—, —B—B—, R³, ¹, Y², and X are as defined inFormula XIIIA. Preferably, R³ is hydrogen.

In the second step of Scheme 2, the enamine of Formula XII is hydrolyzedto an intermediate diketone product of Formula XI

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII, usinga reaction scheme substantially the same as that described above forconversion of the substrate of Formula VIII to the intermediate ofFormula VII. Preferably, the substrate of Formula XII corresponds toFormula XIIA

and the diketone product corresponds to Formula XIA

in each of which —A—A—, —B—B—, R³, Y¹, Y², and X are as defined inFormula XIIIA. Preferably, R³ is hydrogen.

Further in accordance with reaction scheme 2, the diketone of Formula XIis reacted with an alkali metal alkoxide to form mexrenone or otherproduct corresponding to Formula X,

in each of which —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in FormulaXIII, and R¹ is as defined in Formula V. The process is carried outusing substantially the same reaction scheme that is described above forthe conversion of the compounds of Formula VI to those of Formula V.Preferably, the substrate of Formula XI corresponds to Formula XIA

and the intermediate product corresponds to Formula XA

in each of which —A—A—, —B—B—, R³, Y¹, Y², and X are as defined inFormula XIIIA, and R¹ is as defined in Formula V. Preferably, R³ ishydrogen.

Mexrenone and other compounds of Formula X are next 9α-hydroxylated by anovel bioconversion process to yield products of Formula IX

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII, and R¹is as defined in Formula V. Among the organisms that can be used in thishydroxylation step are Nocardia conicruria ATCC 31548, Nocardia aurentiaATCC 12674, Corynespora cassiicola ATCC 16718, Streptomyceshydroscopicus ATCC 27438, Mortierella isabellina ATCC 42613, Beauvriabassiana ATCC 7519, Penicillum purpurogenum ATCC 46581, Hypomyceschrysospermus IMI 109891, Thamnostylum piriforme ATCC 8992,Cunninghamella blakesleeana ATCC 8688a, Cunninghamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Trichothecium roseum ATCC 12543,Epicoccum humicola ATCC 12722, Saccharopolyspora eythrae ATCC 11635,Beauvria bassiana ATCC 13144, Arthrobacter simplex, Bacteriumcyclooxydans ATCC 12673, Cylindrocarpon radicicola ATCC 11011, Nocardiaaurentia ATCC 12674, Norcardia restrictus ATCC 14887, Pseudomonastestosteroni ATCC 11996, Rhodococcus equi ATCC 21329, Mycobacteriumfortuitum NRRL B8119, and Rhodococcus rhodochrous ATCC 19150. Thereaction is carried out substantially in the manner described above inconnection with FIGS. 1 and 2. The process of FIG. 1 is particularlypreferred.

Growth media useful in the bioconversions preferably contain betweenabout 0.05% and about 5% by weight available nitrogen; between about0.5% and about 5% by weight glucose; between about 0.25% and about 2.5%by weight of a yeast derivative; and between about 0.05% and about 0.5%by weight available phosphorus. Particularly preferred growth mediainclude the following:

soybean meal: between about 0.5% and about 3% by weight glucose; betweenabout 0.1% and about 1% by weight soybean meal; between about 0.05% andabout 0.5% by weight alkali metal halide; between about 0.05% and about0.5% by weight of a yeast derivative such as autolyzed yeast or yeastextract; between about 0.05% and about 0.5% by weight of a phosphatesalt such as K₂HPO₄; pH=7;

peptone-yeast extract-glucose: between about 0.2% and about 2% by weightpeptone; between about 0.05% and about 0.5% by weight yeast extract; andbetween about 2% and about 5% by weight glucose;

Mueller-Hinton: between about 10% and about 40% by weight beef infusion;between about 0.35% and about 8.75% by weight casamino acids; betweenabout 0.15% and about 0.7% by weight starch.

Fungi can be grown in soybean meal or peptone nutrients, whileactinomycetes and eubacteria can be grown in soybean meal (plus 0.5% to1% by weight carboxylic acid salt such as sodium formate forbiotransformations) or in Mueller-Hinton broth.

The production of 11β-hydroxymexrenone from mexrenone by fermentation isdiscussed in Example 19B. Similar bioconversion processes can be used toprepare other starting materials and intermediates. Example 19Adiscloses the bioconversion of androstendione to11β-hydroxyandrostendione. Example 19C discloses the bioconversion ofmexrenone to 11α-hydroxymexrenone, Δ^(1,2)-mexrenone,6β-hydroxymexrenone, 12β-hydroxymexrenone, and 9α-hydroxymexrenone.Example 19D discloses the bioconversion of canrenone toΔ^(9,11)-canrenone.

The products of Formula IX are novel compounds, which may be separatedby filtration, washed with a suitable organic solvent, e.g., ethylacetate, and recrystallized from the same or a similar solvent. Theyhave substantial value as intermediates for the preparation of compoundsof Formula I, and especially of Formula IA. Preferably, the compounds ofFormula IX correspond to Formula IXA in which —A—A— and —B—B— are—CH₂—CH₂—, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹together constitute the 20-spiroxane ring:

In the next step of the Scheme 2 synthesis, the product of Formula IX isreacted with a dehydration reagent (suitable dehydration reagents suchas PhSOCl or ClSO₃H are known to persons skilled in the art) to producea compound of Formula II

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII, andR¹ is as defined in Formula V. Preferably, the substrate of Formula IXcorresponds to Formula IXA

and the intermediate product corresponds to Formula IIA

in each of which —A—A—, —B—B—, R³, Y¹, Y² and X are as defined inFormula XIIIA, and R¹ is as defined in Formula V. Preferably, R³ ishydrogen.

In the final step of this synthesis scheme, the product of Formula II isconverted to that of Formula I by epoxidation in accordance with themethod described in U.S. Pat. No. 4,559,332; or preferably by the novelepoxidation method of the invention as described hereinabove.

In a particularly preferred embodiment, the overall process of Scheme 2proceeds as follows:

The synthesis in this case begins with a substrate corresponding toFormula XX

where —A—A— and R³ are as defined in Formula XIII, —B—B— is as definedin Formula XIII except that neither R⁶ nor R⁷ is part of a ring fused tothe D ring at the 16,17 positions, and R²⁶ is lower alkyl, preferablymethyl. Preferably, R³ is hydrogen. Reaction of the substrate of FormulaXX with a sulfonium ylide produces the epoxide intermediatecorresponding to Formula XIX

wherein —A—A—, —B—B—, R³ and R²⁶ are as defined in Formula XX.Preferably, R³ is hydrogen.

In the next step of synthesis scheme 3, the intermediate of Formula XIXis converted to a further intermediate of Formula XVIII

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. Preferably, R³is hydrogen. In this step, Formula XIX substrate is converted to FormulaXVIII intermediate by reaction with NaCH(COOEt)₂ in the presence of abase in a solvent.

Exposure of the compound of Formula XVIII to heat, water and an alkalihalide produces a decarboxylated intermediate compound corresponding toFormula XVII

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. Preferably, R³is hydrogen. The process for conversion of the compound of Formula XX tothe compound of Formula XVII corresponds essentially to that describedin U.S. Pat. Nos. 3,897,417, 3,413,288 and 3,300,489, which areexpressly incorporated herein by reference. While the substrates differ,the reagents, mechanisms and conditions for introduction of the17-spirolactone moiety are essentially the same.

Reaction of the intermediate of Formula XVII with a dehydrogenationreagent yields the further intermediate of Formula XVI.

where —A—A—, —B—B— and R³ are as defined in Formula XX. Preferably, R³is hydrogen.

Typically useful dehydrogenation reagents includedichlorodicyanobenzoquinone (DDQ) and chloranil(2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, the dehydrogenationcould be achieved by a sequential halogenation at the 6-position carbonfollowed by dehydrohalogenation reaction.

The intermediate of Formula XVI is next converted to the enamine ofFormula XVB

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. Preferably, R³is hydrogen. Conversion is by cyanidation essentially in the mannerdescribed above for the conversion of the 11α-hydroxy compound ofFormula VIII to the enamine of Formula VII. Typically, the cyanide ionsource may be an alkali metal cyanide. The base is preferablypyrrolidine and/or tetramethylguanidine. A methanol solvent may be used.

The products of Formula XVB are novel compounds, which may be isolatedby chromatography. These and other novel compounds of Formula XV havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Compounds of Formula XVcorrespond to the structure

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII. In themost preferred compounds of Formula XV and Formula XVB, —A—A— and —B—B—are —CH₂—CH₂—, and R³ is hydrogen.

In accordance with the hydrolysis described above for producing thediketone compounds of Formula VI, the enamines of Formula XVB may beconverted to the diketones of Formula XIVB

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. Preferably, R³hydrogen. Particularly preferred for the synthesis of epoxymexrenone arethose compounds of Formula XIV which also fall within the scope ofFormula XIVB as defined below.

The products of Formula XIVB are novel compounds, which may be isolatedby precipitation. These and other novel compounds of Formula XIV havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Compounds of Formula XIVcorrespond to the structure

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII. In themost preferred compounds of Formula XIV and XIVB, —A—A— and —B—B— are—CH₂—CH₂—, and R³ is hydrogen.

The compounds of Formula XIVB are further converted to compounds ofFormula XXXI using essentially the process described above forconverting the diketone of Formula VI to the hydroxyester of Formula V.In this instance, it is necessary to isolate the intermediate XXXI

before further conversion to a product of Formula XXXII

wherein —A—A—, —B—B— and R³ are as defined in Formula XX, and R¹ is asdefined in Formula V. Preferably, R³ is hydrogen. Preferred compounds ofFormula XXXI are those which fall within Formula IIA. The compounds ofFormula XXXI are converted to compounds of Formula XXXII using themethod described hereinabove or in U.S. Pat. No. 4,559,332.

Preferably, the compound of Formula XIV is4′S(4′α),7′α-1′,2′,3′,4,4′,5,5′,6′,7′,8′,10′,12′,13′,14′,15′,16′-hexadecahydro-10β-,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]-cyclopenta[a]phenanthrene]5′-carbonitrile;and the compound of Formula XV is5′R(5′α),7′β-20′-amino-1′,2′,3′,4′,5′,6′,7′,8′,10′,12′,13′,14′,15′,16′-tetradecahydro-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]-cyclopenta[a]phenanthrene]-5′-carbonitrile.In a particularly preferred embodiment, the overall process of Scheme 3proceeds as follows:

The first three steps of Scheme 4 are the same as those of Scheme 3,i.e., preparation of an intermediate of Formula XVII starting with acompound corresponding to Formula XX.

Thereafter, the intermediate of Formula XVII is epoxidized, for example,using the process of U.S. Pat. No. 4,559,332 to produce the compound ofFormula XXIV

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. However, in aparticularly preferred embodiment of the invention, the substrate ofFormula XVII is epoxidized across the 9,11-double bond using anoxidation reagent comprising an amide type peroxide activator, mostpreferably trichloroacetamide, according to the process as describedabove in Scheme 1 for the conversion of the enester of Formula II to theproduct of Formula I. The conditions and proportions of reagents forthis reaction are substantially as described for the conversion of theFormula II enester to epoxymexrenone. Particularly preferred compoundsof Formula XXIV are those in which —A—A— and —B—B— are as defined inFormula XIII and R³ is hydrogen.

It has been found that the epoxidation of the substrate of Formula XVIIcan also be effected in very good yield using a peracid such as, forexample, m-chloroperoxybenzoic acid. However, the trichloroacetamidereagent provides superior results in minimizing the formation ofBayer-Villager oxidation by-product. The latter by-product can beremoved, but this requires trituration from a solvent such as ethylacetate, followed by crystallization from another solvent such asmethylene chloride. The epoxy compound of Formula XXIV is dehydrogenatedto produce a double bond between the 6- and 7-carbons by reaction with adehydrogenation (oxidizing) agent such as DDQ or chloranil, or using thebromination/dehydrobromination (or otherhalogenation/dehydrohalogenation) sequence, to produce another novelintermediate of Formula XXIII

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XX.Particularly preferred compounds of Formula XXIII are those in which—A—A— and —B—B— are as defined in Formula XIII and R³ is hydrogen.

While direct oxidation is effective for the formation of the product ofFormula XXIII, the yields are generally low. Preferably, therefore, theoxidation is carried out in two steps, first halogenating the substrateof Formula XXIV at the C-6 position, then dehydrohalogenating to the6,7-olefin. Halogenation is preferably effected with an N-halo organicreagent such as, for example, N-bromosuccinamide. Bromination is carriedout in a suitable solvent such as, for example, acetonitrile, in thepresence of halogenation promoter such as benzoyl peroxide. The reactionproceeds effectively at a temperature in the range of about 50° to about100° C., conveniently at atmospheric reflux temperature in a solventsuch as carbon tetrachloride, acetonitrile or mixture thereof. However,reaction from 4 to 10 hours is typically required for completion of thereaction. The reaction solvent is stripped off, and the residue taken upin a water-immiscible solvent, e.g., ethyl acetate. The resultingsolution is washed sequentially with a mild alkaline solution (such asan alkali metal bicarbonate) and water, or preferably saturated brine tominimize product losses, after which the solvent is stripped and a theresidue taken up in another solvent (such as dimethylformamide) that issuitable for the dehydrohalogenation reaction.

A suitable dehydrohalogenation reagent, e.g.,1,4-diazabicyclo[2,2,2]octane (DABCO) is added to the solution, alongwith an alkali metal halide such as LiBr, the solution heated to asuitable reaction temperature, e.g., 60° to 80° C., and reactioncontinued for several hours, typically 4 to 15 hours, to complete thedehydrobromination. Additional dehydrobromination reagent may be addedas necessary during the reaction cycle, to drive the reaction tocompletion. The product of Formula XXIII may then be recovered, e.g., byadding water to precipitate the product which is then separated byfiltration and preferably washed with additional amounts of water. Theproduct is preferably recrystallized, for example fromdimethylformamide.

The products of Formula XXIII, such as 9,11-epoxycanrenone, are novelcompounds, which may be isolated by extraction/crystallization. Theyhave substantial value as intermediates for the preparation of compoundsof Formula I, and especially of Formula IA. For example, they may beused as substrates for the preparation of compounds of Formula XXII.

Using substantially the process described above for the preparation ofcompounds of Formula VII, the compounds of Formula XXIII are reactedwith cyanide ion to produce novel epoxyenamine compounds correspondingto Formula XXII

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XX.Particularly preferred compounds of Formula XXII are those in which—A—A— and —B—B— are as defined in Formula XIII and R³ is hydrogen.

The products of Formula XXII are novel compounds, which may be isolatedby precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. In the most preferred compounds of FormulaXXII, —A—A— and —B—B— are —CH₂—CH₂—, and R³ is hydrogen.

Using substantially the process described above for preparation ofcompounds of Formula VI, the epoxyenamine compounds of Formula XXII areconverted to novel epoxydiketone compounds of Formula XXI

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula XIII. Inthe most preferred compounds of Formula XXI, —A—A— and —B—B— are—CH₂—CH₂— and R³ is hydrogen.

The products of Formula XXI are novel compounds, which may be isolatedby precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXI are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXI, —A—A— and —B—B— are—CH₂—CH₂— and R₃ is hydrogen.

Using substantially the process described above for preparation of thehydroxyester compounds of Formula V from the diketone compounds ofFormula VI, the epoxydiketone compounds of Formula XXI are converted tocompounds of Formula XXXII

wherein —A—A—, —B—B— and R³ are as defined in Formula XX, and R¹ is asdefined in Formula V.

As in the conversion of the diketone of formula V to the hydroxyester offormula VI, a 5-β-cyano-7-ester intermediate is also formed in theconversion of the epoxydiketone of formula XXI to compounds of formulaXXXII. The 5-β-cyano-7-ester intermediates in both series can beisolated by treatment of the corresponding diketone with an alcohol suchas methanol in the presence of a base such as triethylamine. Preferably,the intermediates are prepared by refluxing a mixture of the diketone inan alcohol such as methanol containing about 0.1 to about 2 equivalentsof triethylamine per mole of diketone for about 4 to about 16 hours. Theproducts are isolated in pure form by cooling the mixture to about 25degrees followed by filtration. The isolated intermediates can beconverted to the compounds of Formula XXXII on treatment with a basesuch an alkali metal alkoxide in a solvent, preferably an alcohol suchas methanol. Use of an alkoxide in an alcohol establishes an equilibriummixture similar to that formed when the corresponding diketone ofFormula XXI is treated under the same conditions.

In addition, the 7β-ester of the compound of Formula XXXII (for example7-methyl hydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7β,21-dicarboxylate,γ-lactone) has been observed by chromatography in the crude product ofthe final step of the process of Scheme 4. Alkoxide and/or cyanide inthe solution reacts with and converts the 7α-ester into an epimericmixture of the 7α-ester and its 7β-ester epimer. The pure 7β-ester canbe isolated from the epimeric mixture by selective crystallization.

Preferably, the compound of Formula XXI is4′S(4′α),7′α-9′,11α-epoxyhexadecahydro-10β-,13′β-dimethyl-3′5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]-cyclopenta[a]phenanthrene-5′-carbonitrile;the compound of Formula XXII is5′R(5′α),7′β-20′-amino-9,11β-epoxyhexadecahydro-10′,13′-dimethyl-3′,5-dioxospiro[furan-2(3H),17′a(5′H)[7,4]methene[4H]cyclopenta[a]phenanthrene-5′-carbonitrile; and thecompound of Formula XXIII is9,11α-epoxy-17α-hydroxy-3-oxopregna-4,6-diene-21-carboxylic acid,γ-lactone.

In a particularly preferred embodiment, the overall process of Scheme 4proceeds as follows:

The process of scheme 5 begins with a substrate corresponding to FormulaXXIX

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. The followingmicroorganisms are capable of carrying out the 9α-hydroxylation of acompound of Formula XXXV (such as androstendione)

wherein —A—A—, —B—B— and R³ are as defined in Formula XIII, to acompound of Formula XXIX under conditions similar to those described inExample 19B:

Asperigillus niger ATCC 16888 and 26693, Corynespora cassiicola ATCC16718, Curvularia clavata ATCC 22921, Mycobacterium fortuitum NRRLB8119, Nocardia canicruria ATCC 31548, Pycnosporium spp. ATCC 12231,Stysanus microsporus ATCC 2833, Syncephalastrum racemosum ATCC 18192,and Thamnostylum piriforme ATCC 8992.

The substrate corresponding to Formula XXIX is converted to a product ofFormula XXVIII

by reaction with trimethylorthoformate, wherein —A—A—, —B—B— and R³ areas defined in Formula XX. Following the formation of the compounds ofFormula XXVIII, those compounds are converted to the compounds ofFormula XXVII using the method described above for conversion of the

substrate of Formula XX to Formula XVII. Compounds of Formula XXVII havethe structure:

wherein —A—A—, —B—B— and R³ are as defined in Formula XX, and R^(x) isany of the common hydroxy protecting groups. Alternatively, the C9α-hydroxy group can be protected at an earlier step in this synthesisscheme if protection at that step is desired, i.e., the C9 hydroxy ofthe compound of Formula XXVIII or the C9 hydroxy of the compound ofFormula XXIX can be protected with any of the common hydroxy protectinggroups.

Using the method described above for the preparation of compounds ofFormula XVI, compounds of Formula XXVII are oxidized to yield novelcompounds corresponding to Formula XXVI

wherein —A—A—, —B—B— and R³ are as defined in Formula XX. Particularlypreferred compounds of Formulae XXIX, XXVIII, XXVII and XXVI are thosein which —A—A— and —B—B— are as defined in Formula XIII, and R³ ishydrogen.

The products of Formula XXVI are novel compounds, which may be isolatedby precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXVI are those in which —A—A— and —B—B— are as defined in Formula XIII,and R³ is hydrogen. In the most preferred compounds of Formula XXVI, and—A—A— and —B—B— are —CH₂—CH₂—, and R³ is hydrogen.

Using the method defined above for cyanidation of compounds of FormulaVIII, the novel intermediates of Formula XXVI are converted to the novel9-hydroxyenamine intermediates of Formula XXV

wherein —A—A—, —B—B— and R³ are as defined in Formula XX.

The products of Formula XXV are novel compounds, which may be isolatedby precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXV are those in which —A—A— and —B—B— are as defined in Formula XIII,and R³ is hydrogen. In the most preferred compounds of Formula XXVI, and—A—A— and —B—B— are —CH₂—CH₂—, and R³ is hydrogen.

Using essentially the conditions described above for the preparation ofthe diketone compounds of Formula VI, the 9-hydroxyenamine intermediatesof Formula XXV are converted to the diketone compounds of Formula XIVB.Note that in this instance the reaction is effective for simultaneoushydrolysis of the enamine structure and dehydration at the 9,11positions to introduce the 9,11 double bond. The compound of Formula XIVis then converted to the compound of Formula XXXI, and thence to thecompound of Formula XIII, using the same steps that are described abovein scheme 3.

Preferably, the compound of Formula XIV is4′S(4′α),7′α-1′,2′,3′,4,4′,5,5′,6′,7′,8′,10′,12′,13′,14′,15′,16′-hexadecahydro-10β-,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]-cyclopenta[a]phenanthrene]5′-carbonitrile;the compound of Formula XXV is5′R(5′α),7′β-20′-aminohexadecahydro-9′β-hydroxy-10′a,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile;the compound of Formula XXVI is9α,17α-dihydroxy-3-oxopregna-4,6-diene-21-carboxylic acid, γ-lactone;and the compound of Formula XXVII is9α,17α-dihydroxy-3-oxopregn-4-ene-21-carboxylic acid, γ-lactone.

In a particularly preferred embodiment, the overall process of Scheme 5proceeds as follows:

Scheme 6 provides an advantageous method for the preparation ofepoxymexrenone and other compounds corresponding to Formula I, startingwith 11α or 11β-hydroxylation of androstendione or other compound ofFormula XXXV

wherein —A—A—, —B—B— and R³ are as defined in Formula XIII, producing anintermediate corresponding to the Formula XXXVI or its corresponding11β-hydroxy isomer

where —A—A—, —B—B— and R³ are as defined in Formula XIII. Except for theselection of substrate, the process for conducting the 11α-hydroxylationis essentially as described hereinabove for Scheme 1. The followingmicroorganisms are capable of carrying out the 11α-hydroxylation ofandrostendione or other compound of Formula XXXV:

Absidia glauca ATCC 22752, Aspergillus flavipes ATCC 1030, Aspergillusfoetidus ATCC 10254, Aspergillus fumigatus ATCC 26934, Aspergillusochraceus NRRL 405 (ATCC 18500), Aspergillus niger ATCC 11394,Aspergillus nidulans ATCC 11267, Beauveria bassiana ATCC 7159, Fusariumoxysporum ATCC 7601, Fusarium oxysporum cepae ATCC 11171, Fusarium liniATCC IFO 7156, Gibberella fujikori ATCC 14842, Hypomyces chyrsospermusIMI 109891, Mycobaterium fortuitum NRRL B8119, Penicillum patulum ATCC24550, Pycnosporium spp. ATCC 12231, Rhizopus arrhizus ATCC 11145,Saccharopolyspora erythraea ATCC 11635, Thamnostylum piriforme ATCC8992, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b, andTrichothecium roseum ATCC 12519 and ATCC 8685.

The following microorganisms are capable of carrying out the11β-hydroxylation of androstendione or other compound of Formula XXXV:

Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC 16888 and ATCC26693, Epicoccum oryzae ATCC 7156, Curvularia lunata ATCC 12017,Cunninghamella blakesleeana ATCC 8688a, and Pithomyces atro-olivaceousIFO 6651.

11α-Hydroxyandrost-4-ene-3,17-dione, or other compound of Formula XXXVI,is next converted to 11α-hydroxy-3,4-enol ether of Formula (101):

where —A—A—, —B—B— and R³ are as defined in Formula XIII and R¹¹ ismethyl or other lower alkyl (C₁ to C₄), by reaction with an etherifyingreagent such as trialkyl orthoformate in the presence of an acidcatalyst To carry out this conversion, the 11α-hydroxy substrate isacidified by mixing with an acid such as, e.g., benzene sulfonic acidhydrate or toluene sulfonic acid hydrate and dissolved in a loweralcohol solvent, preferably ethanol. A trialkyl orthoformate, preferablytriethyl orthoformate is introduced gradually over a period of 5 to 40minutes while maintaining the mixture in the cold, preferably at about0° C. to about 15° C. The mixture is then warmed and the reactioncarried out at a temperature of between 20° C. and about 60° C.Preferably the reaction is carried out at 30° to 50° C. for 1 to 3hours, then heated to reflux for an additional period, typically 2 to 6hours, to complete the reaction. Reaction mixture is cooled, preferablyto 0° to 15°, preferably about 5° C., and the solvent removed undervacuum.

Using the same reaction scheme as described in Scheme 3, above, for theconversion of the compound of Formula XX to the compound of FormulaXVII, a 17-spirolactone moiety of Formula XXXIII is introduced into thecompound of Formula 101. For example, the Formula 101 substrate may bereacted with a sulfonium ylide in the presence of a base such as analkali metal hydroxide in a suitable solvent such as DMSO, to produce anintermediate corresponding to Formula 102:

where —A—A—, R³, R¹¹, and —B—B— are as defined in Formula 101. Theintermediate of Formula 102 is then reacted with a malonic acid diesterin the presence of an alkali metal alkoxide to form the five memberedspirolactone ring and produce the intermediate of Formula 103

where —A—A—, R³, R¹¹ and —B—B— are as defined in Formula 102, and R¹² isa C₁ to C₄ alkyl, preferably ethyl. Finally, the compound of Formula 103in a suitable solvent, such as dimethylformamide, is subjected to heatin the presence of an alkali metal halide, splitting off thealkoxycarbonyl moiety and producing the intermediate of Formula 104:

where again —A—A—, R³, R¹¹ and —B—B— are as defined in Formula 102.

Next the 3,4-enol ether compound 104 is converted to the compound ofFormula XXIII, i.e., the compound of Formula VIII in which R⁸ and R⁹together form the moiety of Formula XXXIII. This oxidation step iscarried out in essentially the same manner as the oxidation step forconversion of the compound of Formula XXIV to the intermediate ofFormula XXIII in the synthesis of Scheme 4. Direct oxidation can beeffected using a reagent such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) ortetrachlorobenzoquinone (chloranil), or preferably a two stage oxidationis effected by first brominating, e.g., with an N-halo brominating agentsuch as N-bromosuccinamide (NBS) or 1,3-dibromo-5,5-dimethyl hydantoin(DBDMH) and then dehydrobrominating with a base, for example with DABCOin the presence of LiBr and heat. Where NBS is used for bromination, anacid must also be employed to convert 3-enol ether to the enone. DBDMH,an ionic rather than free radical bromination reagent, is effective byitself for bromination and conversion of the enol ether to the enone.

The compound of Formula VIII is then converted to epoxymexrenone orother compound of Formula I by the steps described hereinabove forScheme 1.

Each of the intermediates of Formulae 101, 102, 103 and 104 is a novelcompound having substantial value as an intermediate for epoxymexrenoneor other compounds of Formulae IA and I. In each of the compounds ofFormulae 101, 102, 103 and 104, —A—A— and —B—B— are preferably —CH₂—CH₂—and R³ is hydrogen, lower alkyl or lower alkoxy. Preferably, R³ ishydrogen. Most preferably, the compound of Formula 101 is3-ethoxy-11α-hydroxyandrost-3,5-dien-17-one, the compound of Formula 102is 3-ethoxyspiro[androst-3,5-diene-17β,2′-oxiran]-11α-ol, the compoundof Formula 103 is ethyl hydrogen3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-dicarboxylate,gamma-lactone, and the compound of Formula 104 is3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid,gamma-lactone.

In a particularly preferred embodiment, the overall process of Scheme 6proceeds as follows:

It is hypothesized that epoxymexrenone and other compounds correspondingto Formula I likewise can be prepared from 11β-hydroxyandrostendione orother compounds of Formula XXXV which have been 11β-hydroxylated. Inother words, epoxymexrenone and other compounds corresponding to FormulaI can be prepared in accordance with the general process set forth inScheme 6 using either an α-hydroxylated substrate of Formula XXXV or thecorresponding β-hydroxylated substrate.

Scheme 7

Scheme 7 provides for the synthesis of epoxymexrenone and othercompounds of Formula I using a starting substrate comprisingβ-sitosterol, cholesterol, stigmasterol or other compound of FormulaXXXVII

where —A—A—, R³, and —B—B— are as defined in Formula XIII; D—D is—CH₂—CH₂— or —CH═CH—; and each of R¹³, R¹⁴, R¹⁵ and R¹⁶ is independentlyselected from among hydrogen or C₁ to C₄ alkyl. R³ is preferablyhydrogen.

In the first step of the synthesis 11α-hydroxyandrostendione or othercompound of Formula XXXVI is prepared by bioconversion of the compoundof Formula XXXVII. The bioconversion process is carried outsubstantially in accordance with the method described hereinabove forthe 11α-hydroxylation of canrenone (or other substrate of Formula XIII).

In the synthesis 11α-hydroxyandrostendione, 4-androstene-3,17-dione isinitially prepared by bioconversion of the compound of Formula XXXVII.This initial bioconversion may be carried out in the manner described inU.S. Pat. No. 3,759,791, which is expressly incorporated herein byreference. Thereafter, 4-androstene-3,17-dione is converted to11α-hydroxyandrostenedione substantially in accordance with the methoddescribed hereinabove for the 11α-hydroxylation of canrenone (or othersubstrate of Formula XIII).

The remainder of the synthesis of Scheme 7 is identical to Scheme 6. Ina particularly preferred embodiment, the overall process of Scheme 7proceeds as follows:

It is hypothesized that epoxymexrenone and other compounds correspondingto Formula I likewise can be prepared in accordance with the generalprocess set forth in Scheme 7 when the product of the bioconversion ofβ-sitosterol or other compounds of Formula XXXVIII is11β-hydroxyandrostendione or other compounds of Formula XXXV which havebeen 11β-hydroxylated. In other words, epoxymexrenone and othercompounds corresponding to Formula I can be prepared in accordance withthe general process set forth in Scheme 7 when the bioconversion ofβ-sitosterol or other compounds of Formula XXXVIII results in thepreparation of either an α-hydroxylated substrate of Formula XXXV or thecorresponding β-hydroxylated substrate.

Scheme 8

A significant complication in the synthesis of epoxymexrenone andrelated compounds is the need for stereoselective introduction of anα-alkoxycarbonyl substituent at the 7-carbon, without unwantedmodifications at other sites of the steroidal structure. In accordancewith the invention, it has been discovered that an effective synthesispath for introduction of a 7α-alkoxycarbonyl substituent involves thefollowing steps: (i) initial cyanidation at the 7-carbon of the steroid,(ii) hydrolysis of the 7-cyano steroid to form a mixture of7α-carboxylic acid and 7β-carboxylic acid steroids, (iii) formation of a5,7-lactone steroid from the 7α-carboxylic acid steroid, and (iv)separation of the 7β-carboxylic acid steroid from the 5,7-lactonesteroid. A base-mediated opening reaction of the 5,7-lactone steroidwith an alkylating reagent produces the desired 7α-alkoxycarbonylsteroid.

Accordingly, the process of Scheme 8 is generally directed to a processfor the preparation of a 3-keto-7α-alkoxycarbonyl substitutedΔ^(4,5)-steroid comprising reacting an alkylating reagent with a3-keto-4,5-dihydro-5,7-lactone steroid substrate in the presence of abase. The lactone substrate is substituted with keto at the 3-carbon,and further comprises the moiety:

where C(5) represents the 5-carbon and C(7) represents the 7-carbon ofthe steroid structure of the substrate. Conversion of the 5,7-lactone tothe 7α-alkoxycarbonyl is preferably effected by reaction with an alkylhalide in the presence of the base. The alkyl halide reagent ispreferably an iodide, most preferably methyl iodide.

Further in accordance with the invention, an advantageous process hasbeen discovered for the preparation of the 4,5-dihydro-5,7-lactonesteroid compound described above. In this process, a3-keto-Δ^(4,5)-7α-cyano substituted steroid substrate is converted tothe 7-carboxylic acid, and the acid in turn reacts with the trialkylorthoformate in an acidified lower alcohol solvent to yield the5,7-lactone. Reaction with orthoformate esters also converts the 3-ketogroup to the 3-acyclic or cyclic ketal 5,7-lactone (the lactone isunderstood to form first). Preferably, the 3-ketal 5,7-lactone is a3-dialkyl ketal 5,7-lactone. More preferably, the alkyl moiety of thealcohol solvent is the same as the alkyl moiety of the orthoformatealkoxy groups (and most preferably all are methyl) because: the alkoxymoieties of the ketal can derive either from the orthoformate or thealcohol; mixed ketals are not preferred; and 3-dimethoxy is preferred.Where the ketal is an ethylene ketal, the alkyl moiety of the alcoholsolvent need not be the same as the alkyl moiety of the orthoformatealkoxy groups. The 3-ketal-5,7-lactone is readily hydrolyzed to the3-keto-5,7-lactone, a crystalline compound which can be easily purified.Since only the 7α-carboxylic acid undergoes the lactonization reaction,complete stereospecificity is realized. The 7β-acid may then be removedfrom the reaction mixture in the form of its salt, e.g., by treating the7β-acid with a mild base such as sodium bicarbonate.

The 7-cyano substrate for the formation of the 5,7-lactone can beprepared in a known manner. For example, a substrate unsubstituted atthe 7-carbon may be reacted with a slight excess of cyanide ion,preferably about 1.05 to about 1.25 equivalents per equivalent substratein a weakly acidic solution comprising a water/DMSO solvent mixture.Preferably, the reaction mixture includes a carboxylic acid, e.g., aboutone equivalent acetic acid per equivalent substrate. Both the 7α- and7β-CN isomers are formed with the 7α-isomer as the major isomer. The7α-cyano steroid may be recovered in a conventional manner. Othermethods known to the art are useful in this ancillary preparation.

Generally in accordance with Scheme 8, the 5,7-lactone may be formedfrom a 7-carboxy intermediate (which itself is prepared by hydrolyzing a7-cyano intermediate) that is substituted at the 17-position with eitherketo, R⁸ or R⁹, where R⁸ and R⁹ are as described above, and havingeither an aliphatic, olefin, epoxide or hydroxy substitutedconfiguration at C-9 and C-11, i.e.,

where —A—A—, —B—B— and R³ are as defined above, R⁸⁰ and R⁹⁰ are the sameas R⁸ and R⁹, or R⁸⁰ and R⁹⁰ together constitute keto, and R¹⁸ is asdescribed below regarding Scheme 9, and —E—E— is selected from among

The compound of Formula XLII is then converted to the7α-alkoxy-carbonyl:

In each of XL, XLI, XLII and XLVIII, R⁸⁰ and R⁹⁰ together preferablycomprise keto or

where Y¹, Y², X and C(17) are as defined above, and most preferably R⁸⁰and R⁹⁰ together comprise

R³ is preferably H, R¹ is preferably methoxycarbonyl, and —A—A— and—B—B— are each preferably —CH₂—CH₂—. It will be understood that thereactions can also be carried out with the 3-keto group protected byconverting it to and maintaining it in each ether or ketal formthroughout the reaction sequence. Alternative processes of Scheme 8comprise use of various intermediates within the scope of Formulae XLIand XLII as recited hereinabove.

Noting that the reagent for formation of the 5,7-lactone from the3-keto-Δ^(4,5)-7-carboxylic acid per Scheme 8 is the trialkylorthoformate, the same reagent used in conversion of the11α-hydroxyandrostendione to the 3-enol ether-3,5-diene-11α-hydroxyintermediate 101 of Scheme 6, it is believed that the path of the Scheme8 reaction is dependent on substitution at C-7. Reaction withorthoformate in the presence of H⁺ forms an intermediate carbonium ionhaving a carboxyl at C-7 and its positive charge in equilibrium betweenC-3 and C-5. Upon loss of the proton, the C-3 carbonium ion yields thecompound of Formula 101, while the C-5 carbonium ion yields the lactone.With hydrogen at C-7, it is believed that 3,5-dien-3-alkoxy (enol ether)is favored because of the double bond conjugation. With the 7α-CO₂substituent at C-7, the C-5 carbonium ion is captured by the carboxy andthe 5,7-lactone is formed. At this point the 3-keto group ispreferentially converted to the ketal, thereby driving the reaction tocompletion.

Preferred embodiments of Scheme 8 are described in Schemes 9 and 10,infra.

Scheme 9

Scheme 9 begins with the same substrate as Scheme 4, i.e., the compoundof Formula XX. This substrate is first oxidized to the compound ofFormula B:

where —A—A—, R³, and —B—B— are as defined in Formula XIII. The oxidationreaction is conducted in accordance with any of the reaction schemesdescribed above for conversion of the compound of Formula XXIV to theintermediate of Formula XXIII in the synthesis of Scheme 4. Using themethods described for Scheme 8, the compound of Formula B is convertedto the 7-cyano intermediate of Formula C:

where —A—A—, R³, and —B—B— are as defined in Formula XIII. Next, thecompound of Formula C is converted to the 5,7-lactone of Formula D:

where —A—A—, R³, and —B—B— are as defined in Formula XIII and R¹⁷ isC₁-C₄ alkyl, using the trialkyl orthoformate reagent utilized previouslyin Scheme 6. The 5,7-lactone of Formula D is readily separated from theunreacted 7-β-COOH, e.g., by removal of the acid via a bicarbonate wash,thereby establishing the desired C-7 stereochemistry and impedingepimerization in subsequent reactions that are conducted under basicconditions. Esterification of the lactone per reaction with alkylhalide, as described in Scheme 8, yields the enester intermediate ofFormula II.

Continuing the Scheme 9 synthesis, the compound of Formula D isconverted to a compound of Formula II. With the 3-keto group protectedby having been converted to the ketal, a 20-spiroxane group of FormulaXXXIII is selectively introduced at the 17-position in accordance withthe reaction scheme described above for Schemes 3 and 6, supra, therebyproducing a compound of Formula E

Because the 3-ketone is protected, hydrolysis conditions may be selectedwhich are optimal for attacking the 17-ketone without concern for theformation of by-products through reaction at the 3-position. Afterhydrolysis of the 3-ketal compound of Formula E to the 3-keto groupstructure of Formula F

the latter intermediate is reacted with alkyl iodide in the presence ofbase, per the conversion of Scheme 8, to produce the intermediateenester of Formula II. Finally, the latter intermediate is converted toepoxymexrenone or other compound of Formula I, using any of the methodsdescribed above for Scheme 1.

Scheme 9 benefits not only from the control of stereochemistry affordedby the 5,7-lactone intermediate, but enjoys the further advantage ofallowing for a wider range of hydrolysis conditions without interferenceof the 17-spirolactone.

Like the reactions for other synthesis schemes of this invention, thereactions of Scheme 9 may be used for conversion of substrates otherthan those particularly described above. Thus, for example, theconversion of 3-keto- or 3-ketal-7-cyano steroids to 3-keto- or3-ketal-5,7-lactone, or the conversion of the 3-keto- or3-ketal-5,7-lactone to 7α-alkoxycarbonyl, may be carried out oncompounds substituted at the 17-carbon by R⁸ and R⁹ as defined above, ormore particularly by a substituent of Formula:

where X, Y¹ and Y² are as defined above and C(17) indicates the17-carbon. However, important advantages are realized, especially inprocess economics, by use of the specific reaction sequence using17-keto substrates and following the specific reaction scheme describedabove for introduction of 17-spirolactone and 7α-alkoxycarbonyl into a3-keto-Δ^(9,11) steroid.

The lactones of Formula D, E, and F are novel compounds which are usefulin the preparation of epoxymexrenone and other compounds of Formula Iand IA in accordance with the synthesis of Scheme 9. In these compounds—A—A— and —B—B— are preferably —CH₂—CH₂— and R³ is hydrogen, lower alkylor lower alkoxy. Most preferably the compound of Formula D is where R¹⁷is methoxy.

In a particularly preferred embodiment, the overall process of Scheme 9proceeds as follows:

Scheme 10 is the same as Scheme 9 through the formation of the 7-cyanointermediate of Formula C. In the next step of Scheme 10, 7-cyanosteroid is reacted with trialkyl orthoformate in an alkanol solvent,preferably trimethyl orthoformate in methanol, to simultaneously protectthe 3-keto and 17-keto groups, by converting the former to the enolether and the latter to the ketal. Thereafter the 7-cyano group isreduced to 7-formyl, e.g., by reaction with a dialkyl aluminum hydride,preferably diisobutyl aluminum hydride, thereby producing a compound ofFormula 203:

where —A—A—, R³, and —B—B— are as defined in Formula XIII, and R¹⁸ isC₁-C₄ alkyl. Prior protection of the keto groups, as described above,prevents their reduction by the dialkyl aluminum hydride. Theintermediate of Formula 203 is next reacted with dilute aqueous acid toselectively hydrolyze the 17-ketal, in the presence of excess alcohol(R¹⁹OH), producing the intermediate of Formula 204:

where R¹⁹ is selected from among lower alkyl (preferably C₁ to C₄), orthe R¹⁹ groups at the 3-position forming a cyclic O,O-oxyalkyleneoxysubstituent at the 3-carbon. The hemiacetal [204] is further protectedby treatment with alkanol (R¹⁹OH) in the presence of non-aqueous acid toproduce the intermediate of Formula 205:

where —A—A—, —B—B—, R³ and R¹⁹ are as defined above, and R²⁰ is C₁ to C₄alkyl. The 17-spirolactone moiety can then be introduced in accordancewith the reaction steps described above for Schemes 3 and 6, thusproceeding through the sequence outlined below:

wherein —A—A—, —B—B—, R³, R¹⁹, and R²⁰ are as defined above and R²⁵ isC₁ to C₄ alkyl.

Thereafter the 3-position is deprotected by conventional hydrolysis toreintroduce the 3-keto group and 5,7-hemiacetal, producing the furtherintermediate corresponding to Formula 209:

where —A—A—, —B—B— and R³ are as defined above. Next, a 9,11 epoxidemoiety is introduced in accordance with any of the methods describedabove for conversion of the compounds of Formula II to the compounds ofFormula I. Under the oxidizing conditions of the epoxidation reaction,the hemiacetal partially converts to the 5,7-lactone, thereby producinga further intermediate corresponding to Formula 211

where —A—A—, —B—B— and R³ are as defined above. Any remaining9,11-epoxy-5,7-hemiacetal intermediate reaction product of Formula 210:

wherein —A—A—, —B—B— and R³ are as defined, is readily oxidized byconventional means to the compound of Formula 211. Finally, theintermediate of Formula 211 is converted to epoxymexrenone or othercompound of Formula I using the method described in Scheme 8 for theconversion of the 5,7-lactone to the 7α-alkoxycarbonyl compound. Thus,overall, Scheme 10 proceeds as follows, it being understood that atleast the following steps may be carried out in situ without recovery ofthe intermediate. Overall, the synthesis of Scheme 10 proceeds asfollows:

As in the case of Scheme 9 the reactions described above for Scheme 10offer important advantages, especially with regard to process economics;but the novel reactions of Scheme 10 also have more generic applicationto substrates other than those particularly described. For example,introduction of the 7-formyl group into a 3-enol ether steroid,protection of the resulting 7-formyl-Δ-5,6-3,4-enol ether, hydrolysis tothe 5,7-hemiacetal, and subsequent deprotection can be conducted onsteroids substituted at the 17-position by R⁸ and R⁹ as defined above,or more particularly by a substituent of Formula:

where X, Y¹, Y², and C(17) are as defined above.

Alternative processes of Scheme 10 comprise use of the variousintermediates within the scope of Formulae A203 through A210,respectively, hereinabove. Each of the intermediates of Formulae A203through A211 is a novel compound which is useful in the preparation ofepoxymexrenone and other compounds of Formula I and IA in accordancewith the synthesis of Scheme 10.

In a particularly preferred embodiment, the overall process of Scheme 10proceeds as follows:

From the several schemes that are illustrated above, it will beunderstood that the reaction steps selected for use in the processes ofthe invention provide substantial flexibility in the manufacture ofepoxymexrenone and related compounds. The key features include, interalia: (a) bioconversion of a substrate such as canrenone,androstendione, or β-sitosterol to an 11α- or 9α-hydroxy derivative(with simultaneous conversion of β-sitosterol to a 17-keto structure;(b) introduction of the 9,11 double bond by dehydration of a compoundcontaining either an 11α- or 9α-hydroxy group, followed by introductionof the epoxy group by oxidation of the 9,11 double bond; (c) attachmentof a 7α-alkoxycarbonyl by formation of the enamine, hydrolysis of theenamine to the diketone, and reaction of the diketone with an alkalimetal alkoxide; (d) formation of the 20-spiroxane ring at the 17position; (e) formation of the 5,7-lactone, and esterification of thelactone to the 7-alkoxycarbonyl; (f) protection of the 3-ketone byconversion to 3-enol ether or 3-ketal during various of the conversionsat other positions (including formation of the 20-spiroxane ring at the17-position. With few limitations, these four component process elements(b) to (d) can be conducted in almost any sequence. Process elements (e)and (f) offer comparable flexibility. They provide a route toepoxymexrenone and other compounds of Formula I which are muchsimplified as compared to the process of U.S. Pat. No. 4,559,332.Moreover, they provide important benefits in productivity and yield.

In the descriptions of the reaction schemes as set forth above,recovery, isolation and purification of reaction products can generallybe carried out by methods well known to those skilled in the art. Exceptwhere otherwise indicated, conditions, solvents, and reagents are eitherconventional, not narrowly critical, or both. However, certain of thespecific procedures as particularly described above provide advantageswhich contribute to the favorable overall yield and/or productivity ofthe various process steps and process schemes, and/or to high quality ofthe intermediates and ultimate 9,11-epoxy steroid products.

The utility of 20-Spiroxane compounds produced in accordance with theinvention is described in Grob U.S. Pat. No. 4,559,332 which isexpressly incorporated herein by reference.

20-Spiroxane compounds produced in accordance with the invention aredistinguished by favorable biological properties and are, therefore,valuable pharmaceutical active ingredients. For example, they have astrong aldosterone-antagonistic action in that they reduce and normalizeunduly high sodium retention and potassium excretion caused byaldosterone. They therefore have, as potassium-saving diuretics, animportant therapeutic application, for example in the treatment ofhypertension, cardiac insufficiency or cirrhosis of the liver.

20-Spiroxane derivatives having an aldosterone-antagonistic action areknown, cf., for example, Fieser and Fieser: Steroids; page 708 (ReinholdPubl. Corp., New York, 1959) and British Patent Specification No.1,041,534; also known are analogously active 17β-hydroxy-21-carboxylicacids and their salts, cf., for example, U.S. Pat. No. 3,849,404.Compounds of this kind that have hitherto been used in therapy, however,have a considerable disadvantage in that they always possess a certainsexual-specific activity which has troublesome consequences sooner orlater in the customary long-term therapy. Especially undesirable are thetroublesome effects that can be attributed to the anti-androgenicactivity of the known anti-aldosterone preparations.

The methods, processes and compositions of the invention, and theconditions and reagents used therein, are further described in thefollowing examples.

EXAMPLE 1

Slants were prepared with a growth medium as set forth in Table 1

TABLE 1 Y P D A (medium for slants and plates) yeast extract 20 gpeptone 20 g glucose 20 g agar 20 g distilled water, q.s. to 1000 ml pHas is 6.7 adjust at pH 5 with H₃PO₄ 10% w/v Distribute for slants: 7.5ml in 180 × 18 mm tubes for plates (10 cm of φ) 25 ml in 200 × 20 mmtubes sterilize at 120° C. for 20 minutes pH after sterilization: 5

To produce first generation cultures, a colony of Aspergillus ochraceuswas suspended in distilled water (2 ml) in a test tube; and 0.15 mlaliquots of this suspension applied to each of the slants that had beenprepared as described above. The slants were incubated for seven days at25° C., after which the appearance of the surface culture was that of awhite cottony mycelium. The reverse was pigmented in orange in the lowerpart, in yellow-orange in the upper part.

The first generation slant cultures were suspended in a sterile solution(4 ml) containing Tween 80 nonionic surfactant (3% by weight), and 0.15ml aliquots of this suspension were used to inoculate second generationslants that had been prepared with the growth medium set forth in Table2

TABLE 2 (for second generation and routine slants) malt extract 20 gpeptone  1 g glucose 20 g agar 20 g distilled water q.s. to 1000 ml pHas is 5.3 distribute in tubes (180 × 18 mm) ml 7.5 sterilize at 120° C.for 20 minutes

The second generation slants were incubated for 10 days at 25° C.,producing a heavy mass of golden-colored spores; reverse pigmented inbrown orange.

A protective medium was prepared having the composition set forth inTable 3.

TABLE 3 PROTECTIVE MEDIUM Skim milk 10 g distilled water 100 ml In a 250ml flask containing 100 ml of distilled water at 50° C., add skim milk.Sterilize at 120° C. for 15 minutes. Cool at 33° C. and use before theday is over

Cultures from five of the second generation slants were suspended in theprotective solution (15 ml) in a 100 ml flask. The suspension wasdistributed in aliquots (0.5 ml each) among 100×10 mm tubes forlyophilization. These were pre-frozen at −70° to −80° C. in anacetone/dry ice bath for 20 minutes, then transferred immediately to adrying room pre-cooled to −40° to −50° C. The pre-frozen aliquots werelyophilized at a residual pressure of 50μ Hg and ≦−30° C. At the end ofthe lyophilization, two to three granules of sterile silica gel wereadded to each tube with moisture indicator and flame seal.

To obtain mother culture slants suitable for industrial scalefermentation, a single aliquot of lyophilized culture, which had beenprepared in the manner described above, was suspended in distilled water(1 ml) and 0.15 ml aliquots of the suspension were used to inoculateslants that had been provided with a growth medium having thecomposition set forth in Table 2. The mother slants were incubated forseven days at 25° C. At the end of incubation, the culture developed onthe slants was preserved at 4° C.

To prepare a routine slant culture, the culture from a mother slant wassuspended in a sterile solution (4 ml) containing Tween 80 (3% byweight) and the resulting suspension distributed in 0.15 ml aliquotsamong slants which had been coated with the growth medium described inTable 2. The routine slant cultures may be used to inoculate the primaryseed flasks for laboratory or industrial fermentations.

To prepare a primary seed flask culture, the culture from a routineslant, which had been prepared as described above, was removed andsuspended in a solution (10 ml) containing Tween 80 (3% by weight). A0.1 aliquot of the resulting suspension was introduced into a 500 mlbaffled flask containing a growth medium having the composition setforth in Table 4.

TABLE 4 (for primary and transformation flask culture and round bottomedflask) glucose 20 g peptone 20 g yeast autolysate 20 g distilled waterq.s to pH as is 5.2 adjust at pH 5.8 with NaOH 20% distribute in 500 mlbaffled flask 100 ml distribute in 2000 ml round bottomed flasks (3baffles) 500 ml sterilize 120° C. × 20 min. pH after sterilization about5.7

The seed flask was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 24 hours at 28° C., thereby producing a culture in theform of pellet-like mycelia having diameters of 3 to 4 mm. Onmicroscopic observation, the seed culture was found to be a pureculture, with synnematic growth, with big hyphae and well twisted. ThepH of the suspension was 5.4 to 5.6. PMV was 5 to 8% as determined bycentrifugation (3000 rpm×5 min.).

A transformation flask culture was prepared by inoculating a growthmedium (100 ml) having the composition set forth Table 4 in a second 500ml shaker flask with biomass (1 ml) from the seed culture flask. Theresulting mixture was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 18 hours at 28° C. The culture was examined and foundto comprise pellet like mycelia with a 3-4 mm diameter. On microscopicexamination, the culture was determined to be a pure culture, withsynnematic and filamentous growth in which the apical cells were full ofcytoplasm and the olden cells were little vacuolated. The pH of theculture suspension was 5 to 5.2 and the PMV was determined bycentrifugation to be between 10% and 15%. Accordingly, the culture wasdeemed suitable for transformation of canrenone to 11α-hydroxycanrenone.

Canrenone (1 g) was micronized to about 5μ and suspended in sterilewater (20 ml). To this suspension were added: a 40% (w/v) sterileglucose solution; a 16% (w/v) sterile solution of autolyzed yeast; and asterile antibiotic solution; all in the proportions indicated for 0hours reaction time in Table 5. The antibiotic solution had beenprepared by dissolving kanamicyn sulfate (40 mg), tetracycline HCl (40mg) and cefalexin (200 mg) in water (100 ml). The steroid suspension,glucose solution, and autolyzed yeast solution were added gradually tothe culture contained in the shaker flask.

TABLE 5 Indicative Additions of Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in Shake Flaskyeast Steroid auto- anti- Reaction Suspension glucose lised biotic timeapprox. solution sol. solution hours ml mg. ml ml. ml 0 1 50 1 0.5 1 8 2100 2 1 24 2 100 1 0.5 1 32 5 250 2 1 48 2 100 1 0.5 1 56 5 250 2 1 72 3150 1 0.5 1 90

As reaction proceeded, the reaction mixture was periodically analyzed todetermine glucose content, and by thin layer chromatography to determineconversion to 11α-hydroxycanrenone. Additional canrenone substrate andnutrients were added to the fermentation reaction mixture during thereaction at rates controlled to maintain the glucose content in therange of about 0.1% by weight. The addition schedule for steroidsuspension, glucose solution, autolyzed yeast solution and antibioticsolution is set forth in Table 5. The transformation reaction continuedfor 96 hours at 25° C. on a rotary shaker (200 rpm and 5 cmdisplacement). The pH ranged between 4.5 and 6 during the fermentation.Whenever the PMV rose to or above 60%, a 10 ml portion of broth culturewas withdrawn and replaced with 10 ml distilled water. The disappearanceof canrenone and appearance of 11α-hydroxycanrenone were monitoredduring the reaction by sampling the broth at intervals of 4, 7, 23, 31,47, 55, 71, 80, and 96 hours after the start of the fermentation cycle,and analyzing the sample by TLC. The progress of the reaction asdetermined from these samples is set forth in Table 6

TABLE 6 Time Course of Bioconversion of Canrenone in Shake FlaskTransformation Ratio Time Canrenone Rf. 11αhydroxy Canrenone hours RF. =0.81 RF. = 0.29 0 100 0.0 4 50 50 7 20 80 23 20 80 31 30 70 47 20 80 5530 70 71 25 75 80 15 85 96 ˜10 ˜90

EXAMPLE 2

A primary seed flask culture was prepared in the manner described inExample 1. A nutrient mixture was prepared having the composition setforth in Table 7

TABLE 7 For Transformation Culture in 10 l glass fermenter quantity g/lglucose  80 g 20 peptone  80 g 20 yeast autolyzed  80 g 20 antifoam SAG471 0.5 g deionized water q.s. to 4 l sterilize the empty fermenter for30 minutes at 130° C. load it with 3 l of deionized water, heat at 40°C. add while stirring the components of the medium stir for 15 minutes,bring to volume of 3.9 l pH as is 5.1 adjust of 5.8 with NaOH 20% w/vsterilize at 120° C. × 20 minutes pH after sterilization 5.5-5.7

An initial charge of this nutrient mixture (4 L) was introduced into atransformation fermenter of 10 L geometric volume. The fermenter was ofcylindrical configuration with a height to diameter ratio of 2.58. Itwas provided with a 400 rpm turbine agitator having two No. 2 diskwheels with 6 blades each. The external diameter of the impellers was 80mm, each of the blades was 25 mm in radial dimension and 30 mm high, theupper wheel was positioned 280 mm below the top of the vessel, the lowerwheel was 365 mm below the top, and baffles for the vessel were 210 mmhigh and extended radially inwardly 25 mm from the interior verticalwall of the vessel.

Seed culture (40 ml) was mixed with the nutrient charge in thefermenter, and a transformation culture established by incubation for 22hours at 28° C., and an aeration rate of 0.5 l/1-min. at a pressure of0.5 kg/cm². At 22 hours, the PMV of the culture was 20-25% and the pH 5to 5.2.

A suspension was prepared comprising canrenone (80 g) in sterile water(400 ml), and a 10 ml portion added to the mixture in the transformationfermenter. At the same time a 40% (w/v) sterile glucose solution, a 16%(w/v) sterile solution of autolyzed yeast, and a sterile antibioticsolution were added in the proportions indicated in Table 8 at 0 hoursreaction time. The antibiotic solution was prepared in the mannerdescribed in Example 1.

TABLE 8 Indicative Additions of Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in 10 1 GlassFermenter yeast anti- Reaction Steroid glucose autolyzed biotic timeSuspension approx solution solution solution hours ml gr ml ml ml 0 10 425 12.5 40 4 25 12.5 8 10 4 25 12.5 12 25 12.5 16 10 4 25 12.5 20 2512.5 24 10 4 25 12.5 40 28 10 4 25 12.5 32 12.5 5 25 12.5 36 12.5 5 2512.5 40 12.5 5 25 12.5 44 12.5 5 25 12.5 48 12.5 5 25 12.5 40 52 12.5 525 12.5 56 12.5 5 25 12.5 60 12.5 5 25 12.5 64 12.5 5 25 12.5 68 12.5 525 12.5 72 12.5 5 25 12.5 40 76 12.5 5 25 12.5 80 84 88

As reaction proceeded, the reaction mixture was periodically analyzed todetermine glucose content, and by thin layer chromatography to determineconversion to 11α-hydroxycanrenone. Based on TLC analysis of reactionbroth samples as described hereinbelow, additional canrenone was addedto the reaction mixture as canrenone substrate was consumed. Glucoselevels were also monitored and, whenever glucose concentration droppedto about 0.05% by weight or below, supplemental glucose solution wasadded to bring the concentration up to about 0.25% by weight. Nutrientsand antibiotics were also added at discrete times during the reactioncycle. The addition schedule for steroid suspension, glucose solution,autolyzed yeast solution and antibiotic solution is set forth in Table8. The transformation reaction continued for 90 hours at an aerationrate of 0.5 vol. air per vol. liquid per minute (vvm) at a positive headpressure of 0.3 kg/cm². The temperature was maintained at 28° C. untilPVM reached 45%, then decreased to 26° C. and maintained at thattemperature as PVM grew from 45% to 60%, and thereafter controlled at24° C. The initial agitation rate was 400 rpm, increasing to 700 rpmafter 40 hours. The pH was maintained at between 4.7 and 5.3 byadditions of 2M orthophosphoric acid or 2M NaOH, as indicated. Foamingwas controlled by adding a few drops of Antifoam SAG 471 as foamdeveloped. The disappearance of canrenone and appearance of11α-hydroxycanrenone were monitored at 4 hour intervals during thereaction by TLC analysis of broth samples. When most of the canrenonehad disappeared from the broth, additional increments were added.

After all canrenone additions had been made, the reaction was terminatedwhen TLC analysis showed that the concentration of canrenone substraterelative to 11α-hydroxycanrenone product had dropped to about 5%.

At the conclusion of the reaction cycle, the fermentation broth wasfiltered through cheese cloth for separation of the mycelium from theliquid broth. The mycelia fraction was resuspended in ethyl acetateusing about 65 volumes (5.2 liters) per gram canrenone charged over thecourse of the reaction. The suspension of mycelia in ethyl acetate wasrefluxed for one hour under agitation, cooled to about 20° C., andfiltered on a Buchner. The mycelia cake was washed sequentially withethyl acetate (5 vol. per g canrenone charge; 0.4 L) and deionized water(500 ml) to displace the ethyl acetate extract from the cake. The filtercake was discarded. The rich extract, solvent washing and water washingwere collected in a separator, then allowed to stand for 2 hours forphase separation.

The aqueous phase was then discarded and the organic phase concentratedunder vacuum to a residual volume of 350 ml. The still bottoms werecooled to 15° C. and kept under agitation for about one hour. Theresulting suspension was filtered to remove the crystalline product, andthe filter cake was washed with ethyl acetate (40 ml). After drying, theyield of 11α-hydroxycanrenone was determined to be 60 g.

EXAMPLE 3

A spore suspension was prepared from a routine slant in the mannerdescribed in Example 1. In a 2000 ml baffled round bottomed flask (3baffles, each 50 mm×30 mm), an aliquot (0.5 ml) of the spore suspensionwas introduced into a nutrient solution (500 ml) having the compositionset forth in Table 4. The resulting mixture was incubated in the flaskfor 24 hours at 25° C. on an alternating shaker (120 strokes per min.;displacement 5 cm), thereby producing a culture which, on microscopicexamination, was observed to appear as a pure culture with hyphae welltwisted. The pH of the culture was between about 5.3 and 5.5, and thePMV (as determined by centrifugation at 3000 rpm for 5 min.) was 8 to10%.

Using the culture thus prepared, a seed culture was prepared in astainless steel fermenter of vertical cylindrical configuration, havinga geometric volume of 160 L and an aspect ratio of 2.31 (height=985 mm;diameter=425 mm). The fermenter was provided with a disk turbine typeagitator having two wheels, each wheel having six blades with anexternal diameter of 240 mm, each blade having a radial dimension of 80mm and a height of 50 mm. The upper wheel was positioned at a depth of780 mm from the top of the fermenter, and the second at a depth of 995mm. Vertical baffles having a height of 890 mm extended radiallyinwardly 40 mm from the interior vertical wall of the fermenter. Theagitator was operated at 170 rpm. A nutrient mixture (100 L) having thecomposition set forth in Table 9 was introduced into the fermenter,followed by a portion of preinoculum (1 L) prepared as described aboveand having a pH of 5.7.

TABLE 9 For Vegetative Culture in 160 L Fermenter About 8 L are neededto Seed Productive fermenter Quantity g/L glucose    2 kg 20 peptone   2 kg 20 yeast autolysed    2 kg 20 antifoam SAG 471 0.010 Kg tracesdeionized water q.s. to 100 L sterilize the empty fermenter for 1 hourat 130° C. load it with 6 L of deionized water; heat at 40° C. add whilestirring the components of the medium stir for 15 minutes, bring tovolume of 95 L sterilization at 121° C. for 30 minutes poststerilization pH is 5.7 add sterile deionized water to 100 L

The inoculated mixture was incubated for 22 hours at an aeration rate of0.5 L/L-min. at a head pressure of 0.5 kg/cm². The temperature wascontrolled at 28° C. until PMV reached 25%, and then lowered to 25° C.The pH was controlled in the range of 5.1 to 5.3. Growth of myceliumvolume is shown in Table 10, along with pH and dissolved oxygen profilesof the seed culture reaction.

TABLE 10 Time Course for Mycelial Growth in Seed Culture Fermentationpacked mycelium volume (pmv) % Fermentation (3000 dissolved period h pHrpms 5 min) oxygen % 0 5.7 ± 0.1 100 4 5.7 ± 0.1 100 8 5.7 ± 0.1 12 ± 385 ± 5 12 5.7 ± 0.1 15 ± 3 72 ± 5 16 5.5 ± 0.1 25 ± 5 40 ± 5 20 5.4 ±0.1 30 ± 5 35 ± 5 22 5.3 ± 0.1 33 ± 5 30 ± 5 24 5.2 ± 0.1 35 ± 5 25 ± 5

Using the seed culture thus produced, a transformation fermentation runwas carried out in a vertical cylindrical stainless steel fermenterhaving a diameter of 1.02 m, a height of 1.5 m and a geometric volume of1.4 m³. The fermenter was provided with a turbine agitator having twoimpellers, one positioned 867 cm below the top of the reactor and theother positioned 1435 cm from the top. Each wheel was provided with sixblades, each 95 cm in radial dimension and 75 cm high. Vertical baffles1440 cm high extended radially inwardly 100 cm from the interiorvertical wall of the reactor. A nutrient mixture was prepared having thecomposition set forth in Table 11

TABLE 11 For Bioconversion Culture in 1000 L Fermenter Quantity g/Lglucose   16 kg 23 peptone   16 kg 23 yeast autolysed   16 kg 23antifoam SAG 471 0.080 Kg traces deionized water q.s. to 700 L sterilizethe empty fermenter for 1 hour at 130° C. load it with 600 L ofdeionized water; heat at 40° C. add while stirring the components of themedium stir for 15 minutes, bring to volume of 650 L sterilization at121° C. for 30 minutes post sterilization pH is 5.7 add steriledeionized water to 700 L

An initial charge (700 L) of this nutrient mixture (pH=5.7) wasintroduced into the fermenter, followed by the seed inoculum of thisexample (7 L) prepared as described above.

The nutrient mixture containing inoculum was incubated for 24 hours atan aeration rate of 0.5 L/L-min at a head pressure of 0.5 kg/cm². Thetemperature was controlled at 28° C., and the agitation rate was 110rpm. Growth of mycelium volume is shown in Table 12, along with pH anddissolved oxygen profiles of the seed culture reaction.

TABLE 12 Time Course for Mycelial Growth in Fermenter of theTransformation Culture packed mycelium volume (pmv) % Fermentation (3000rpmx5 dissolved period h pH min) oxygen % 0 5.6 ± 0.2 100 4 5.5 ± 0.2100 8 5.5 ± 0.2 12 ± 3 95 ± 5 12 15 ± 3 90 ± 5 16 5.4 ± 0.1 20 ± 5 75 ±5 20 5.3 ± 0.1 25 ± 5 60 ± 5 22 5.2 ± 0.1 30 ± 5 40 ± 5

At the conclusion of the incubation, pelleting of the mycelium wasobserved, but the pellets were generally small and relatively looselypacked. Diffuse mycelium was suspended in the broth. Final pH was 5.1 to5.3.

To the transformation culture thus produced was added a suspension ofcanrenone (1.250 kg; micronized to 5μ) in sterile water (5 L). Sterileadditive solution and antibiotic solution were added in the proportionsindicated at reaction time 0 in Table 14. The composition of theadditive solution is set forth in Table 13.

TABLE 13 ADDITIVE SOLUTION (for transformative culture) quantitydextrose   40 Kg yeast autolysate    8 Kg antifoam SAG 471 0.010 Kgdeionized water q.s. to 100 l sterilize a 150 l empty fermenter for 1hour at 130° C. load it with 70 l of deionized water; heat at 40° C. addwhile stirring the components of “additive solution” stir for 30minutes, bring to volume of 95 l pH as is 4.9 sterilize at 120° C. × 20minutes pH after sterilization about 5

Bioconversion was carried out for about 96 hours with aeration at 0.5L/L-min. at a head pressure of 0.5 kg/cm² and a pH of ranging between4.7 and 5.3, adjusted as necessary by additions of 7.5 M NaOH or 4 MH₃PO₄. The agitation rate was initially 100 rpm, increased to 165 rpm at40 hours and 250 rpm at 64 hours. The initial temperature was 28° C.,lowered to 26° C. when PMV reached 45%, and lowered to 24° C. when PMVrose to 60%. SAG 471 in fine drops was added as necessary to controlfoaming. Glucose levels in the fermentation were monitored at 4 hourintervals and, whenever the glucose concentration fell below 1 gpl, anincrement of sterile additive solution (10 L) was added to the batch.Disappearance of canrenone and appearance of 11α-hydroxycanrenone werealso monitored during the reaction by HPLC. When at least 90% of theinitial canrenone charge had been converted to 11α-hydroxycanrenone, anincrement of 1.250 kg canrenone was added. When 90% of the canrenone inthat increment was shown to have been converted, another 1.250 kgincrement was introduced. Using the same criterion further increments(1.250 kg apiece) were added until the total reactor charge (20 kg) hadbeen introduced. After the entire canrenone charge had been delivered tothe reactor, reaction was terminated when the concentration of unreactedcanrenone was 5% relative to the amount of 11α-hydroxycanrenoneproduced. The schedule for addition of canrenone, sterile additivesolution, and antibiotic solution is as shown in Table 14.

TABLE 14 Additions of the Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in FermenterCANRENONE Sterile anti- Reaction in suspension additive biotic volumetime Progress- solution solution liters hours Kg ive Kg liters litersabout 0 1.250 1.25 10 8 700 4 10 8 1.250 2.5 10 12 10 16 1.250 10 20 1024 1.250 5 10 8 800 28 1.250 10 32 1.250 10 36 1.250 10 40 1.250 10 441.250 10 48 1.250 12.5 10 6 900 52 1.250 10 56 1.250 10 60 1.250 10 641.250 10 68 1.250 10 72 1.250 20 10 8 1050 76  0 80 84 88 92 Total

When bioconversion was complete, the mycelia were separated from thebroth by centrifugation in a basket centrifuge. The filtrate wasdetermined by HPLC to contain only 2% of the total quantity of11α-hydroxycanrenone in the harvest broth, and was therefore eliminated.The mycelia were suspended in ethyl acetate (1000 L) in an extractiontank of 2 m³ capacity. This suspension was heated for one hour underagitation and ethyl acetate reflux conditions, then cooled andcentrifuged in a basket centrifuge. The mycelia cake was washed withethyl acetate (200 L) and thereafter discharged. The steroid richsolvent extract was allowed to stand for one hour for separation of thewater phase. The water phase was extracted with a further amount ofethyl acetate solvent (200 L) and then discarded. The combined solventphases were clarified by centrifugation and placed in a concentrator(500 L geometric volume) and concentrated under vacuum to a residualvolume of 100 L. In carrying out the evaporation, the initial charge tothe concentrator of combined extract and wash solutions was 100 L, andthis volume was kept constant by continual or periodic additions ofcombined solution as solvent was taken off. After the evaporation stepwas complete, the still bottoms were cooled to 20° C. and stirred fortwo hours, then filtered on a Buchner filter. The concentrator pot waswashed with ethyl acetate (20 L) and this wash solution was then used towash the cake on the filter. The product was dried under vacuum for 16hours at 50° C. Yield of 11α-hydroxycanrenone was 14 kg.

EXAMPLE 4

Lyophilized spores of Aspergillus ochraceus NRRL 405 were suspended in acorn steep liquor growth medium (2 ml) having the composition set forthin Table 15:

TABLE 15 Corn Steep Liquor Medium (Growth Medium for Primary SeedCultivation) Corn steep liquor 30 g Yeast extract 15 g Ammoniumphosphate  3 g Monobasic Glucose (charge after sterilization) 30 gdistilled water, q.s. to 1000 ml pH as is: 4.6, adjust to pH 6.5 with20% NaOH, distribute 50 ml to 250 ml Erlenmeyer flask sterilize 121° C.for 20 minutes.

The resulting suspension was used in an inoculum for the propagation ofspores on agar plates. Ten agar plates were prepared, each bearing asolid glucose/yeast extract/phosphate/agar growth medium having thecomposition set forth in Table 16:

TABLE 16 GYPA (Glucose/Yeast Extract/Phosphate Agar for Plates) Glucose(charge after sterilization)  10 g Yeast extract 2.5 g K₂HPO₄   3 g Agar 20 g distilled water, q.s. to 1000 ml adjust pH to 6.5 sterilize 121°C. for 30 minutes

A 0.2 ml aliquot of the suspension was transferred onto the surface ofeach plate. The plates were incubated at 25° C. for ten days, afterwhich the spores from all the plates were harvested into a sterilecryogenic protective medium having the composition set forth in Table17:

TABLE 17 GYP/Glycerol (Glucose/Yeast Extract/ Phosphate/Glycerol mediumfor stock vials) Glucose (charge after sterilization)  10 g Yeastextract 2.5 g K₂HPO₄   3 g Glycerol  20 g Distilled water, q.s. to 1000mL Sterilize at 121° C. for 30 minutes

The resulting suspension was divided among twenty vials, with one mlbeing transferred to each vial. These vials constitute a master cellbank that can be drawn on to produce working cell banks for use ingeneration of inoculum for bioconversion of canrenone to11α-hydroxycanrenone. The vials comprising the master cell bank werestored in the vapor phase of a liquid nitrogen freezer at −130° C.

To begin preparation of a working cell bank, the spores from a singlemaster cell bank vial were resuspended in a sterile growth medium (1 ml)having the composition set forth in Table 15. This suspension wasdivided into ten 0.2 ml aliquots and each aliquot used to inoculate anagar plate bearing a solid growth medium having the composition setforth in Table 16. These plates were incubated for ten days at 25° C. Bythe third day of incubation, the underside of the growth medium wasbrown-orange. At the end of the incubation there was heavy production ofgolden colored spores. The spores from each plate were harvested by theprocedure described hereinabove for the preparation of the master cellbank. A total of one hundred vials was prepared, each containing 1 ml ofsuspension. These vials constituted the working cell bank. The workingcell bank vials were also preserved by storage in the vapor phase of aliquid nitrogen freezer at −130° C.

Growth medium (50 ml) having the composition set forth in Table 15 wascharged to a 250 ml Erlenmeyer flask. An aliquot (0.5 ml) of workingcell suspension was introduced into the flask and mixed with the growthmedium. The inoculated mixture was incubated for 24 hours at 25° C. toproduce a primary seed culture having a percent packed mycelial volumeof approximately 45%. Upon visual inspection the culture was found tocomprise pellet-like mycelia of 1 to 2 mm diameter; and upon microscopicobservation it appeared as a pure culture.

Cultivation of a secondary seed culture was initiated by introducing agrowth medium having the composition set forth in Table 15 into a 2.8 LFernbach flask, and inoculating the medium with a portion (10 ml) of theprimary seed culture of this example, the preparation of which was asdescribed above. The inoculated mixture was incubated at 25° C. for 24hours on a rotating shaker (200 rpm, 5 cm displacement). At the end ofthe incubation, the culture exhibited the same properties as describedabove for the primary seed culture, and was suitable for use in atransformation fermentation in which canrenone was bioconverted to11α-hydroxycanrenone.

Transformation was conducted in a Braun E Biostat fermenter configuredas follows:

Capacity: 15 liters with round bottom Height: 53 cm Diameter: 20 cm H/D:2.65 Impellers: 7.46 cm diameter, six paddles 2.2 × 1.4 cm each Impellerspacing: 65.5, 14.5 and 25.5 cm from bottom of tank Baffles: four 1.9 ×48 cm Sparger: 10.1 cm diameter, 21 holes ˜1 mm diameter Temperaturecontrol: provided by means of an external vessel jacket

Canrenone at a concentration of 20 g/L was suspended in deionized water(4 L) and a portion (2 L) of growth medium having the composition setforth in Table 18 was added while the mixture in the fermenter wasstirred at 300 rpm.

TABLE 18 (Growth medium for bioconversion culture in 10 L fermenter)Quantity Amount/L glucose (charge after 160 g 20 g sterilization)peptone 160 g 20 g yeast extract 160 g 20 g antifoam SAF471 4.0 ml 0.5ml Canrenone 160 g 20 g deionized water q.s. to 7.5 L sterilize 121° C.for 30 minutes

The resulting suspension was stirred for 15 minutes, after which thevolume was brought up to 7.5 L with additional deionized water. At thispoint the pH of the suspension was adjusted from 5.2 to 6.5 by additionof 20% by weight NaOH solution, and the suspension was then sterilizedby heating at 121° C. for 30 minutes in the Braun E fermenter. The pHafter sterilization was 6.3±0.2, and the final volume was 7.0 L. Thesterilized suspension was inoculated with a portion (0.5 L) of thesecondary seed culture of this example that has been prepared asdescribed above, and the volume brought up to 8.0 L by addition of 50%sterile glucose solution. Fermentation was carried out at a temperatureof 28° C. until the PMV reached 50%, then lowered to 26° C., and furtherlowered to 24° C. when PMV exceeded 50% in order to maintain aconsistent PMV below about 60%. Air was introduced through the spargerat a rate of 0.5 vvm based on initial liquid volume and the pressure inthe fermenter was maintained at 700 millibar gauge. Agitation began at600 rpm and was increased stepwise to 1000 rpm as needed to keep thedissolved oxygen content above 30% by volume. Glucose concentration wasmonitored. After the initial high glucose concentration fell below 1%due to consumption by the fermentation reaction, supplemental glucosewas provided via a 50% by weight sterile glucose solution to maintainthe concentration in the 0.05% to 1% range throughout the remainder ofthe batch cycle. Prior to inoculation the pH was 6.3±0.2. After the pHdropped to about 5.3 during the initial fermentation period, it wasmaintained in the range of 5.5±0.2 for the remainder of the cycle byaddition of ammonium hydroxide. Foam was controlled by adding apolyethylene glycol antifoam agent sold under the trade designation SAG471 by OSI Specialties, Inc.

Growth of the culture took place primarily during the first 24 hours ofthe cycle, at which time the PMV was about 40%, the pH was about 5.6 andthe dissolved oxygen content was about 50% by volume. Canrenoneconversion began even as the culture was growing. Concentrations ofcanrenone and 11α-hydroxycanrenone were monitored during thebioconversion by analyzing daily samples. Samples were extracted withhot ethyl acetate and the resulting sample solution analyzed by TLC andHPLC. The bioconversion was deemed complete when the residual canrenoneconcentration was about 10% of the initial concentration. Theapproximate conversion time was 110 to 130 hours.

When bioconversion was complete, mycelial biomass was separated from thebroth by centrifugation. The supernatant was extracted with an equalvolume of ethyl acetate, and the aqueous layer discarded. The mycelialfraction was resuspended in ethyl acetate using approximately 65 volumesper g canrenone charged to the fermentation reactor. The mycelialsuspension was refluxed for one hour under agitation, cooled to about20° C., and filtered on a Buchner funnel. The mycelial filter cake waswashed twice with 5 volumes of ethyl acetate per g of canrenone chargedto the fermenter, and then washed with deionized water (1 L) to displacethe residual ethyl acetate. The aqueous extract, rich solvent, solventwashing and water washing were combined. The remaining exhaustedmycelial cake was either discarded or extracted again, depending onanalysis for residual steroids therein. The combined liquid phases wereallowed to settle for two hours. Thereafter, the aqueous phase wasseparated and discarded, and the organic phase concentrated under vacuumuntil the residual volume was approximately 500 ml. The still bottle wasthen cooled to about 15° C. with slow agitation for about one hour. Thecrystalline product was recovered by filtration, and washed with chilledethyl acetate (100 ml). Solvent was removed from the crystals byevaporation, and the crystalline product dried under vacuum at 50° C.

EXAMPLE 5

Lyophilized spores of Aspergillus ochraceus ATCC 18500 were suspended ina corn steep liquor growth medium (2 ml) as described in Example 4. Tenagar plates were prepared, also in the manner of Example 4. The plateswere incubated and harvested as described in Example 4 to provide amaster cell bank. The vials comprising the master cell bank were storedin the vapor phase of a liquid nitrogen freezer at −130° C.

From a vial of the master cell bank, a working cell bank was prepared asdescribed in Example 4, and stored in the nitrogen freezer at −130° C.

Growth medium (300 mL) having the composition set forth in Table 19 wascharged to a 2 L baffled flask. An aliquot (3 mL) of working cellsuspension was introduced into the flask. The inoculated mixture wasincubated for 20 to 24 hours at 28° C. on a rotating shaker (200 rpm, 5cm displacement) to produce a primary seed culture having a percentpacked mycelial volume of approximately 45%. Upon visual inspection theculture was found to comprise pellet like mycelia of 1 to 2 mm diameter;and upon microscopic observation it appeared as a pure culture.

TABLE 19 Growth medium for primary and secondary seed cultivationAmount/L glucose (charge after 20 g sterilization) peptone 20 g Yeastextract 20 g distilled water q.s. to 1000 mL sterilize 121° C. for 30minutes

Cultivation of a secondary seed culture was initiated by introducing 8 Lgrowth medium having the composition set forth in Table 19 into a 14 Lglass fermenter. Inoculate the fermenter with 160 mL to 200 mL of theprimary seed culture of this example. The preparation of which was asdescribed above.

The inoculated mixture was cultivated at 28° C. for 18-20 hours, 200 rmpagitation, aeration rate was 0.5 vvm. At the end of the propagation, theculture exhibited the same properties as described above for the primaryseed.

Transformation was conducted in a 60 L fermenter, substantially in themanner described in Example 4, except that the growth medium had thecomposition set forth in Table 20, and the initial charge of secondaryseed culture was 350 mL to 700 mL. Agitation rate was initially 200 rpm,but increased to 500 rpm as necessary to maintain dissolved oxygen above10% by volume. The approximate bioconversion time for 20 g/L canrenonewas 80 to 160 hours.

TABLE 20 Growth Medium for Bioconversion Culture in 60 L FermenterQuantity Amount/L glucose (charge after 17.5 g 0.5 g sterilization)peptone 17.5 g 0.5 g yeast extract 17.5 g 0.5 g Canrenone (charge as a 700 g  20 g 20% slurry in sterile water) deionized water, q.s. to 35 Lsterilize 121° C. for 30 minutes

EXAMPLE 6

Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. Using secondary seed culture produced in this manner, twobioconversion runs were made in accordance with a modified process ofthe type illustrated in FIG. 1, and two runs were made with the processillustrated in FIG. 2. The transformation growth medium, canrenoneaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Table 21. Run R2A used a canrenone addition schemebased on the same principle as Example 3, while run R2C modified theExample 3 scheme by making only two additions of canrenone, one at thebeginning of the batch, and one after 24 hours. In runs R2B and R2D, theentire canrenone charge was introduced at the beginning of the batch andthe process generally carried in the manner described in Example 4,except that the canrenone charge was sterilized in a separate vesselbefore it was charged to the fermenter and glucose was added as thebatch progressed. A Waring blender was used to reduce chunks produced onsterilization. In runs R2A and R2B, canrenone was introduced into thebatch in methanol solution, in which respect these runs further differedfrom the runs of Examples 3 and 4, respectively.

TABLE 21 Descriptions of the Initial Canrenone Bioconversion ProcessesRun Number R2A R2B R2C R2D Medium (g/L) Corn steep liq. 30 the same asrun 30 the same as run Yeast extract 15 R2A 15 R2C NH₄H₂PO₄  3  3Glucose 15 30 OSA 0.5 ml 0.5 ml pH adjusted to 6.0 adjusted to with2.5NNaOH 6.5 with 2.5NNaOH Canrenone 10 g/80 ml MEOH 80 g/640 ml MEOHSterilized and Sterilized and added at 0, 18, added at 0 hr all blended;added blended; added 24, 30, 36, 42, at once at: 0 hr: 25 g at: 0 hr:200 g 50, 56, 62 and 24 hr: 200 g 68 hr. Harvest time 143 hrs. 166 hrs.125 hrs. 104 hrs. Bioconversion 45.9% 95.6% 98.1% 95.1%

In runs R2A and R2B, the methanol concentration accumulated to about6.0% in the fermentation beer, which was found to be inhibitory to thegrowth of culture and bioconversion. However, based on the results ofthese runs, it was concluded that methanol or other water-misciblesolvent could serve effectively at lower concentrations to increase thecanrenone charge and provide canrenone as a fine particle precipitateproviding a large interfacial area for supply of canrenone to thesubject to the reaction.

Canrenone proved stable at sterilization temperature (121° C.) butaggregated into chunks. A Waring blender was employed to crush the lumpsinto fine particles, which were successfully converted to product.

EXAMPLE 7

Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. The description and results of Example 7 are shown inTable 22. Using secondary seed culture produced in this manner, onebioconversion (R3C) was carried out substantially as described inExample 3, and three bioconversions were carried out in accordance withthe process generally described in Example 5. In the latter three runs(R3A, R3B and R3D), canrenone was sterilized in a portable tank,together with the growth medium except for glucose. Glucose wasaseptically fed from another tank. The sterilized canrenone suspensionwas introduced into the fermenter either before inoculation or duringthe early stage of bioconversion. In run R3B, supplemental sterilecanrenone and growth medium was introduced at 46.5 hours. Lumps ofcanrenone formed on sterilization were delumped through a Waring blenderthus producing a fine particulate suspension entering the fermenter. Thetransformation growth media, canrenone addition schedules, nutrientaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Tables 22 and 23.

TABLE 22 Descriptions of Process for Canrenone Bioconversion Run NumberR3A R3B R3C R3D Medium (g/L) Corn steep liq. 30 the same as run Peptone:20 the same as run Yeast extract 15 R3A Yeast Ext.: 20 R3A NH₄H₂PO₄  3Glucose: 20 Glucose 15 OSA: 3 ml OSA 0.5 ml pH adjusted to 6.5 adjustedto 6.5 with 2.5NNaOH with 2.5NNaOH Canrenone canrenone was the same asrun Non-sterile The same as run charge at sterilized and R3A canrenone:R3A blended. BI: 50 g BI: 50 g charged by the 16.5 hrs: 110 g 16.5 hrs:110 g scheduled listed BI: 50 g 46.5 hrs: 80 g in Table 23 16.5 hrs: 110g Feedings see Table 23 see Table 23 see Table 23 see Table 23 Harvesttime 118.5 hrs. 118.5 hrs 118.5 hrs 73.5 hrs. Bioconversion 93.7% 94.7%60.0% 68.0%

TABLE 23 The Feeding Schedule for Canrenone, Glucose and Growth Mediumin the Development Experiment R3C Peptone Antibiotics R3A R3B R3D &Yeast 20 mg kana- Canrenone/ Canrenone/ Canrenone/ ext. mycin 20 mgGrowth Growth Growth canrenone Glucose 20 g tetracycline Medium MediumMedium 200 g/2 L 50% each in 100 mg see Table see Table see TableAddition sterile solution IL water cefalexin in 22 22 22 Time hr. DI g gg 50 ml g/L g/L g/L 0 — — — — 50 g/0.4 L 50 g/0.4 L 50 g/0.4 L 14.5 16100 25 50 ml — — — 16.5 — — — — 110 g/1.2 L 110 g/1.2 L 110 g/1.2 L 20.516 140 25 — — — — 28.5 16 140 25 — — — — 34.5 16 150 25 — — — — 40.5 16150 25 50 ml — — — 46.5 880 130 25 — — 80 g/0.8 L — 52.5 160 120 25 — —— — 58.5 160 150 25 — — — — 64.5 160 180 25 50 ml — — — 70.5 160 140 25— — — —

Due to filamentous growth, a highly viscous fermenter broth was seen inall four of the runs of this Example. To overcome obstacles which highviscosity created with respect to aeration, mixing, pH control andtemperature control, the aeration rate and agitation speed wereincreased during these runs. Conversions proceeded satisfactorily underthe more severe conditions, but a dense cake formed above the liquidbroth surface. Some unreacted canrenone was carried out of the broth bythis cake.

EXAMPLE 8

The description and results of Example 8 are summarized in Table 24.Four fermentation runs were made in which 11α-hydroxycanrenone wasproduced by bioconversion of canrenone. In two of these runs (R4A andR4D), the bioconversion was conducted in substantially the same manneras runs R3A and R3D of Example 6. In run R4C, canrenone was converted to11α-hydroxycanrenone generally in the manner described in Example 3. InRun R4B, the process was carried out generally as described in Example4, i.e., with sterilization of canrenone and growth medium in thefermenter just prior to inoculation, all nitrogen and phosphorusnutrients were introduced at the start of the batch, and a supplementalsolution containing glucose only was fed into the fermenter to maintainthe glucose level as the batch proceeded. In the latter process (runR4B), glucose concentration was monitored every 6 hours and glucosesolution added as indicated to control glucose levels in the 0.5 to 1%range. The canrenone addition schedules for these runs are set forth inTable 25.

TABLE 24 Descriptions of the Process Development Experiment of CanrenoneBioconversions Run Number R4A R4B R4C R4D Medium (g/L) Corn steep liq.30 the same as run peptone: 20 the same as run Yeast extract 15 R4AYeast ext.: 20 R4A NH4H2PO4  3 Glucose: 20 Glucose 15 OSA 3 ml OSA 0.5ml pH adjusted to 6.5 adjusted to with 2.5NNaOH 6.5 with 2.5NNaOHCanrenone Canrenone was 160 g canrenone Nonsterile Canrenone was chargeat sterilized and is sterilized canrenone: sterilized and blended. inthe charged by the blended. BI: 40 g fermenter schedule BI: 40 g 23.5hrs: 120 g listed in 23.5 hrs: 120 g Table 25 Medium charge see Table 25see Table 25 see Table 25 see Table 25 Harvest time 122 hrs. 122 hrs.122 hrs. 122 hrs. Bioconversion 95.6% 97.6% 95.4% 96.7%

TABLE 25 The Feeding Schedule of Canrenone, Glucose and Growth Medium inthe Development Experiment R4C Antibiotics Peptone 20 mg kanamycin &Yeast 20 mg tetra- R4A R4B R4D Canrenone ext. 20 g cycline 100 mg GrowthGrowth Growth 200 g /2 L Glucose each in cefalexin in 50 Medium MediumMedium sterile 50% 1 L ml (added in see see see Addition water solutionwater canrenone Table Table Table Time hr. g g g slurry) 24 24 24 14 600135 25 50 ml — — — 20 — 100 — — — — — 23 — — — — 120 g/ — 120 g/ 1.2 L1.2 L 26 — 100 25 — — — — 32 — 135 25 — — — — 38 500 120 25 50 ml — — —44 — 100 25 — — — — 50 — 100 25 — — — — 56 — 150 25 — — — — 62 500 15025 50 ml — — — 68 — 200 25 — — — — 74 — 300 25 — — — — 8- — 100 25 — — —— 86 — 175 25 — — — — 92 — 175 25 — — — — 98 — 150 — — — — — 104 — 175 —— — — — 110 — 175 — — — — — 116 — 200 — — — — —

All fermenters were run under high agitation and aeration during most ofthe fermentation cycle because the fermentation beer had become highlyviscous within a day or so after inoculation.

EXAMPLE 9

The transformation growth media, canrenone addition schedules, harvesttimes, and degrees of conversion for the runs of this Example are setforth in Table 26.

Four bioconversion runs were carried out substantially in the mannerdescribed for run R4B of Example 8, except as described below. In runR5B, the top turbine disk impeller used for agitation in the other runswas replaced with a downward pumping marine is impeller. The downwardpumping action axially poured the broth into the center of the fermenterand reduced cake formation. Methanol (200 ml) was added immediatelyafter inoculation in run R5D. Since canrenone was sterilized in thefermenter, all nutrients except glucose were added at the start of thebatch, obviating the need for chain feeding of sources of nitrogen,sources of phosphorus or antibiotics.

TABLE 26 Process Description of the Process Development Experiment of 10L Scale Bioconversions Run Number R5A R5B R5C R5D Medium (g/L) Cornsteep liq. 30 the same as run Peptone: 20 the same as run Yeast Extract15 R5A Yeast Ext.: 20 R5A NH₄H₂PO₄  3 Glucose: 20 Glucose 15 OSA 3 mlOSA 0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5NNaOH with2.5NNaOH Canrenone charge 160 g canrenone 160 g canrenone 160 gcanrenone 160 g canrenone sterilized in sterilized in sterilized insterilized in the fermenter the fermenter the fermenter the fermenterMedium feeding glucose feeding glucose feeding glucose feeding glucosefeeding Harvest time 119.5 hrs. 119.5 hrs. 106 119.5 hrs. Bioconversion96% 94.1% 88.5% 92.4%

In order to maintain immersion of the solid phase growing above theliquid surface, growth medium (2 L) was added to each fermenter 96 hoursafter the beginning of the batch. Mixing problems were not entirelyovercome by either addition of growth medium or use of a downwardpumping impeller (run R5B) but the results of the runs demonstrated thefeasibility and advantages of the process, and indicated thatsatisfactory mixing could be provided according to conventionalpractices.

EXAMPLE 10

Three bioconversion runs were carried out substantially in the mannerdescribed in Example 9. The transformation growth media, canrenoneaddition schedules, harvest times, and degrees of conversion for theruns of this Example are set forth in Table 27:

TABLE 27 Process Description of the Experiment 10 L Scale BioconversionRun Number R6A R6B R6C Medium (g/L) Corn steep liq. 30 the same as runPeptone: 20 Yeast Extract 15 R6A Yeast Ext.: 20 NH₄H₂PO₄  3 Glucose: 20Glucose 15 OSA OSA 0.5 ml 0.5 ml pH adjusted to 6.5 adjusted to 6.5 withwith 2.5NNaOH 2.5NNaOH Canrenone 160 g 160 g canrenone 160 g canrenonecharge canrenone sterilized in the sterilized in sterilized in thefermenter the fermenter fermenter Medium glucose glucose feeding;glucose feeding feeding; 0.5 L medium and feeding; no 1.3 L medium 0.5 Lsterile other addition and water at 95 hrs 0.8 L sterile water at 71hrs. Harvest time 120 hrs. 120 hrs. 120 hrs. Bioconversion 95% 96% 90%Mass Balance 59% 54% 80%

Growth medium (1.3 L) and sterile water (0.8 L) were added after 71hours in run R6A to submerge mycelial cake which had grown above thesurface of the liquid broth. For the same purpose, growth medium (0.5 L)and sterile water (0.5 L) were added after 95 hours in run R6B. Materialbalance data showed that a better mass balance could be determined wherecake buildup above the liquid surface was minimized.

EXAMPLE 11

Fermentation runs were made to compare pre-sterilization of canrenonewith sterilization of canrenone and growth medium in the transformationfermenter. In run R7A, the process was carried out as illustrated inFIG. 2, under conditions comparable to those of runs R2C, R2D, R3A, R3B,R3D, R4A, and R4D. Run R7B was as illustrated in FIG. 3 under conditionscomparable to those of Examples 4, 9 and 10, and run R4B. Thetransformation growth media, canrenone addition schedules, harvesttimes, and degrees of conversion for the runs of this Example are setforth in Table 28:

TABLE 28 Process Description of the Experiment of 10 L ScaleBioconversions Run Number R7A R7B Medium (g/L) corn steep liq. 30 thesame as run Yeast extract 15 R7A NH₄H₂PO₄  3 Glucose 15 OSA 0.5 ml pHadjusted to 6.5 with 2.5NNaOH Canrenone charge 160 g canrenone 160 gcanrenone was sterilized & was sterilized blended outside in thefermenter the fermenter Medium charge Glucose feeding; Glucose feeding;canrenone was no other added with 1.6 L addition growth medium Harvesttime 118.5 hrs. 118.5 hrs. Bioconversion 93% 89%

A mass balance based on the final sample taken from run R7B was 89.5%,indicating that no significant substrate loss or degradation inbioconversion. Mixing was determined to be adequate for both runs.

Residual glucose concentration was above the desired 5-10 g per litercontrol range during the initial 80 hours. Run performance wasapparently unaffected by a light cake that accumulated in the head spaceof both the fermenters.

EXAMPLE 12

Extraction efficiency was determined in a series of 1 L extraction runsas summarized in Table 29. In each of these runs, steroids wereextracted from the mycelium using ethyl acetate (1 L/L fermentationvolume). Two sequential extractions were performed in each run. Based onRP-HPLC, About 80% of the total steroid was recovered in the firstextraction; and recovery was increased to 95% by the second extraction.A third extraction would have recovered another 3% of steroid. Theremaining 2% is lost in the supernatant aqueous phase. The extract wasdrawn to dryness using vacuum but was not washed with any additionalsolvent. Chasing with solvent would improve recovery from the initialextraction if justified by process economics.

TABLE 29 Recovery of 11α-Hydroxycanrenone at 1 Liter Extraction (% ofTotal) 1st 2nd 3rd Run Number Extract Extract Extract Supernatant R5A79% 16% 2% 2% R5A 84% 12% 2% 2% R4A 72% 20% 4% 4% R4A 79% 14% 2% 5% R4B76% 19% 4% 1% R4B 79% 16% 3% 2% R4B 82% 15% 2% 1% Average 79% 16% 3% 2%

Methyl isobutyl ketone (MIEK) and toluene were evaluated asextraction/crystallization solvents for 11α-hydroxycanrenone at the 1 Lbroth scale. Using the extraction protocol as described hereinabove,both MIBK and toluene were comparable to ethyl acetate in bothextraction efficiency and crystallization performance.

EXAMPLE 13

As part of the evaluation of the processes of FIGS. 2 and 3, particlesize studies were conducted on the canrenone substrate provided at thestart of the fermentation cycle in each of these processes. As describedabove, canrenone fed to the process of FIG. 1 was micronized beforeintroduction into the fermenter. In this process, the canrenone is notsterilized, growth of unwanted microorganisms being controlled byaddition of antibiotics. The processes of FIGS. 2 and 3 sterilize thecanrenone before the reaction. In the process of FIG. 2, this isaccomplished in a blender before introduction of canrenone into thefermenter. In the process of FIG. 3, a suspension of canrenone in growthmedium is sterilized in the fermenter at the start of the batch. Asdiscussed hereinabove, sterilization tends to cause agglomeration ofcanrenone particles. Because of the limited solubility of canrenone inthe aqueous growth medium, the productivity of the process depends onmass transfer from the solid phase, and thus may be expected to dependon the interfacial area presented by the solid particulate substratewhich in turn depends on the particle size distribution. Theseconsiderations initially served as deterrents to the processes of FIGS.2 and 3.

However, agitation in the blender of FIG. 2 and the fermentation tank ofFIG. 3, together with the action of the shear pump used for transfer ofthe batch in FIG. 2, were found to degrade the agglomerates to aparticle size range reasonably approximate that of the unsterilized andmicronized canrenone fed to the process of FIG. 1. This is illustratedby the particle size distributions for the canrenone as available at theoutset of the reaction cycle in each of the three processes. See Table30 and FIGS. 4 and 5.

TABLE 30 Particle Distributions of Three Different Canrenone Samplesmean size Run #: % Sample 45-125 μ <180 μ μ Bioconversion Canrenone  75%   95% — R3C: shipment 93.1% (120 h) R4C: 96.3% (120 h) Blended31.2% 77.2% 139.5 R3A: Sample 94.6% (120 h) R3B: 95.2% (120 h) Sterilize24.7% 65.1% 157.4 R4B: d Sample 97.6% (120 h) R5B: 93.8% (120 h)

From the data in Table 30, it will be noted that agitators and shearpump were effective to reduce the average particle size of thesterilized canrenone to the same order of magnitude as the unsterilizedsubstrate, but a significance size difference remained in favor of theunsterilized substrate. Despite this difference, reaction performancedata showed that the pre-sterilization processes were at least asproductive as the process of FIG. 1. Further advantages may be realizedin the process of FIG. 2 by certain steps for further reducing andcontrolling particle size, e.g., wet milling of sterilized canrenone,and/or by pasteurizing rather than sterilizing.

EXAMPLE 14

A seed culture was prepared in the manner described in Example 5. At 20hours, the mycelia in the inoculum fermenter was pulpy with a 40% PMV.Its pH was 5.4 and 14.8 gpl glucose remained unused.

A transformation growth medium (35 L) was prepared having thecomposition shown in Table 20. In the preparation of feeding medium,glucose and yeast extract were sterilized separately and mixed as asingle feed at an initial concentration of 30% by weight glucose and 10%by weight yeast extract. pH of the feed was adjusted to 5.7.

Using this medium, (Table 20), two bioconversion runs were made for theconversion of canrenone to 11α-hydroxycanrenone. Each of the runs wasconducted in a 60 L fermenter provided with an agitator comprising oneRushton turbine impeller and two Lightnin' A315 impellers.

Initial charge of the growth medium to the fermenter was 35 L.Micronized and unsterilized canrenone was added to an initialconcentration of 0.5%. The medium in the fermenter was inoculated with aseed culture prepared in the manner described in Example 5 at an initialinoculation ratio of 2.5%. Fermentation was carried out at a temperatureof 28° C., an agitation rate of 200 to 500 rpm, an aeration rate of 0.5vvm, and backpressure sufficient to maintain a dissolved oxygen level ofat least 20% by volume. The transformation culture developed during theproduction run was in the form of very small oval pellets (about 1-2mm). Canrenone and supplemental nutrients were chain fed to thefermenter generally in the manner described in Example 1. Nutrientadditions were made every four hours at a ratio of 3.4 g glucose and 0.6g yeast extract per liter of broth in the fermenter.

Set forth in Table 31 are the aeration rate, agitation rate, dissolvedoxygen, PMV, and pH prevailing at stated intervals during each of theruns of this Example, as well as the glucose additions made during thebatch. Table 32 shows the canrenone conversion profile. Run R11A wasterminated after 46 hours; Run R11B continued for 96 hours. In thelatter run, 93% conversion was reached at 81 hours; one more feedaddition was made at 84 hours; and feeding then terminated. Note that asignificant change in viscosity occurred between the time feeding wasstopped and the end of the run.

TABLE 31 air Gluc Time (1 pm) rpm % DO Backpress PMV (%) pH cc (g/l)Fermentation R11A 0.1 20 200 93 0 2 6.17 5.8 7 20 200 85.1 0 5 6.03 5.512.4 20 300 50.2 0 5.43 21.8 20 400 25.5 0 38 6.98 0 29 20 500 17 0 355.22 30.2 20 500 18.8 10 5.01 31 20 500 79 10 4.81 1 35.7 20 500 100 1045 5.57 0 46.2 20 500 23 6 45 5.8 1 Total glucose: 27.5 g/l Total yeastextract: 8.75 g/l Fermentation R11B 0.1 20 200 92.9 0 2 5.98 5.4 7 20200 82.3 0 5 5.9 5 12.4 20 300 49.5 0 5.48 21.8 20 400 18 0 40 7.12 0 2920 500 36.8 0 35 5.1 3 35.7 20 500 94.5 10 4.74 0 46.2 20 500 14.5 6 455.32 2 55 20 500 16.7 10 5.31 0.5 58.6 20 500 19.4 15 5.32 1 61.9 20 50013 15 40 5.36 2 71.7 20 500 13 15 42 5.37 0 81.1 20 500 22.9 15 5.42 2.585.6 20 500 22 15 45 6.47 0 97.5 20 500 108 15 45 6.47 0 117.7 20 500 157.38 0 Total glucose: 63 g/l Total yeast extract: 14.5 g/l

TABLE 32 Concentrations (g/l) Conversion Calc OH-can Conv. rates (g/l/h)Sample Time OH-can Canren. Total (%) (g/l) Calculated MeasuredFermentation R11A: Canrenone conversion R11A-0 0.10 0.00 5.41 5.41R11A-7 7.00 0.18 4.89 5.07 3.58 0.18 0.03 0.03 R11A- 21.80 2.02 2.124.14 48.75 2.44 0.15 0.12 22 R11A- 29.00 3.67 4.14 7.81 47.03 4.48 0.280.23 29 R11A- 35.70 6.68 1.44 8.12 82.27 7.74 0.49 0.45 36 R11A- 46.207.09 0.41 7.51 94.48 8.59 0.08 0.04 46 Fermentation R11B: Canrenoneconversion R11B-0 0.1 0.00 5.60 5.60 R11B-7 7.0 0.20 4.98 5.18 3.78 0.190.03 0.03 R11B- 21.8 2.51 2.46 4.97 50.49 2.52 0.16 0.16 22 R11B- 29.04.48 16.99 21.47 20.87 4.69 0.30 0.27 29 R11B- 35.7 8.18 10.35 18.5344.16 9.70 0.75 0.55 36 R11B- 55.0 17.03 13.20 30.23 56.33 19.50 0.320.36 55 R11B- 58.6 20.80 11.73 32.53 63.95 21.97 0.69 1.05 59 R11B- 61.922.19 8.62 30.81 72.02 24.50 0.77 0.42 62 R11B- 71.7 26.62 3.61 30.2388.06 29.46 0.51 0.45 72 R11B- 81.1 27.13 2.05 29.18 92.97 30.32 0.090.05 81 R11B- 85.6 26.87 2.02 28.88 93.02 30.11 −0.04 −0.06 86 R11B-97.5 23.95 1.71 25.66 93.34 30.22 0.01 −0.25 97 R11B- 117.7 24.10 1.6825.79 93.47 30.26 0.00 0.01 118

EXAMPLE 15

Various cultures were tested for effectiveness in the bioconversion ofcanrenone to 11α-hydroxycanrenone according to the methods generallydescribed above.

A working cell bank of each of Aspergillus niger ATCC 11394, Rhizopusarrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b was prepared inthe manner described in Example 5. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used toinoculate transformation flasks containing the growth medium (30 ml) ofTable 18. Each culture was used for inoculation of two flasks, a totalof six. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36° C.,and a 0.5 ml aliquot of this solution was introduced into each of theflasks. Bioconversion was carried out generally under the conditionsdescribed in Example 5 with additions of 50% by weight glucose solution(1 ml) each day. After the first 72 hours the following observationswere made on the development of mycelia in the respective transformationfermentation flasks:

ATCC 11394—good even growth

ATCC 11145—good growth in first 48 hours, but mycelial clumped into aball; no apparent growth in last 24 hours;

ATCC 6227b—good growth; mycelial mass forming clumped ball.

Samples of the broth were taken to analyze for the extent ofbioconversion. After three days, the fermentation using ATCC 11394provided conversion to 11α-hydroxycanrenone of 80 to 90%; ATCC 11145provided a conversion of 50%; and ATCC 6227b provided a conversion of 80to 90%.

EXAMPLE 16

Using the substantially the method described in Example 15, theadditional microorganisms were tested for effectiveness in theconversion of canrenone to 11α-hydroxycanrenone. The organisms testedand the results of the tests are set forth in Table 33:

TABLE 33 Cultures tested for Bioconversion of canrenone to 11alpha-hydroxy-canrenone approximate Culture ATTC# media¹ resultsconversion Rhizopus oryzae 1145 CSL + 50% — Rhizopus stolonifer 6227bCSL + 80-90% — Aspergillus nidulans 11267 CSL + 50% 80% Aspergillusniger 11394 CSL + 80-90% — Aspergillus ochraceus NRRL CSL + 90% 405Aspergillus ochraceus 18500 CSL + 90% Bacillus subtilis 31028 P&CSL − 0%0% Bacillus subtilis 31028 CSL − 0% 0% Bacillus sp. 31029 P&CSL − 0% 0%Bacillus sp. 31029 CSL − 0% * Bacillus megaterium 14945 P&CSL + 5% 80%*Bacillus megaterium 14945 CSL + 5% 10%* Trichothecium roseum 12519 CSL +80%* 90%* Trichothecium roseum 8685 CSL + 80%* 90%* Streptomyces fradiae10745 CSL + <5% <10% Streptomyces fradiae 10745 TSB − * * Streptomyces13664 CSL − 0% * lavendulae Streptomyces 13664 TSR − 0% 0% lavendulaeNocardiodes simplex 6946 BP − 0% 0% Nocardiodes simplex 13260 BP − * *Pseudomonas sp. 14696 BP − * * Pseudomonas sp. 14696 CSL + <5* <10%Pseudomonas sp. 14696 TSR − 0% * Pseudomonas sp. 13261 BP + * <10%Pseudomonas cruciviae 13262 BP # <10% Pseudomonas putida 15175 BP − 0%0% *formation of other unidentified products ¹Media: CSL - corn steepliquor; TSB - tryptic soy broth; P & CSL - peptone and acorn steepliquor; BP - beef extract and peptone.

EXAMPLE 17

Various microorganisms were tested for effectiveness in the conversionof canrenone to 9α-hydroxycanrenone. Fermentation media for the runs ofthis Example were prepared as set forth in Table 34:

TABLE 34 Soybean Meal: dextrose   20 g soybean meal   5 g NaCl   5 gyeast extract   5 g KH₂PO₄   5 g water to 1 L pH   7.0 Peptone/yeastextract/glucose: glucose   40 g bactopeptone   10 g yeast extract   5 gwater to 1 L Mueller-Hinton: beef infusion  300 g casamino acids 17.5 gstarch  1.5 g water to 1 L

Fungi were grown in soybean meal medium and in peptone-yeast extractglucose; atinomycetes and eubacteria were grown in soybean meal (plus0.9% by weight Na formate for biotransformations) and in Mueller-Hintonbroth.

Starter cultures were inoculated with frozen spore stocks (20 ml soybeanmeal in 250 ml Erlenmayer flask). The flasks were covered with a milkfilter and bioshield. Starter cultures (24 or 48 hours old) were used toinoculate metabolism cultures (also 20 ml in 250 ml Erlenmeyerflask)—with a 10% to 15% crossing volume—and the latter incubated for 24to 48 hours before addition of steroid substrate for the transformationreaction.

Canrenone was dissolved/suspended in methanol (20 mg/ml), filtersterilized, and added to the cultures to a final concentration of 0.1mg/ml. All transformation fermentation flasks were shaken at 250 rpm (2″throw) in a controlled temperature room at 26° C. and 60% humidity.

Biotransformations were harvested at 5 and 48 hours, or at 24 hours,after addition of substrate. Harvesting began with the addition of ethylacetate (23 ml) or methylene chloride to the fermentation flask. Theflasks were then shaken for two minutes and the contents of each flaskpoured into a 50 ml conical tube. To separate the phases, tubes werecentrifuged at 4000 rpm for 20 minutes in a room temperature unit. Theorganic layer from each tube was transferred to a 20 ml borosilicateglass vial and evaporated in a speed vac. Vials were capped and storedat −20° C.

To obtain material for structure determination, biotransformations werescaled up to 500 ml by increasing the number of shake flaskfermentations to 25. At the time of harvest (24 or 48 hours afteraddition of substrate), ethyl acetate was added to each flaskindividually, and the flasks were capped and put back on the shaker for20 minutes. The contents of the flasks were then poured intopolypropylene bottles and centrifuged to separate the phases, or into aseparatory funnel in which phases were allowed to separate by gravity.The organic phase was dried, yielding crude extract of steroidscontained in the reaction mixture.

Reaction product was analyzed first by thin layer chromatography onsilica gel (250 μm) fluorescence backed plates (254 nm). Ethyl acetate(500 μL was added to each vial containing dried ethyl acetate extractfrom the reaction mixture. Further analyses were conducted by highperformance liquid chromatography and mass spectrometry. TLC plates weredeveloped in a 95:5 v/v chloroform/methanol solvent mixture.

Further analysis was conducted by high performance liquid chromatographyand mass spectrometry. A waters HPLC with Millennium software,photodiode array detector and autosampler was used. Reversed phase HPLCused a waters NovaPak C-18 (4 μm particle size) RadialPak 4 mmcartridge. The 25 minute linear solvent gradient began with the columninitialized in water:acetonitrile (75:25), and ended atwater:acetonitrile (25:75). This was followed by a three minute gradientto 100% acetonitrile and 4 minutes of isocratic wash before columnregeneration in initial conditions.

For LC/MS, ammonium acetate was added to both the acetonitrile and waterphases at a concentration of 2 nM. Chromatography was not significantlyaffected. Eluant from the column was split 22:1, with the majority ofthe material directed to the PDA detector. The remaining 4.5% of thematerial was directed to the electrospray ionizing chamber of an SciexAPI III mass spectrometer. Mass spectrometry was accomplished inpositive mode. An analog data line from the PDA detector on the HPLCtransferred a single wave length chromatogram to the mass spectrometerfor coanalysis of the UV and MS data.

Mass spectrometric fragmentation patterns proved useful in sorting fromamong the hydroxylated substrates. The two expected hydroxylatedcanrenones, 11α-hydroxy- and 9α-hydroxy, lost water at differentfrequencies in a consistent manner which could be used as a diagnostic.Also, the 9α-hydroxycanrenone formed an ammonium adduct more readilythan did 11α-hydroxycanrenone. Set forth in Table 35 is a summary of theTLC, HPLC/UV and LC/MS data for canrenone fermentations, showing whichof the tested microorganism were effective in the bioconversion ofcanrenone to 9α-hydroxycanrenone. Of these, the preferred microorganismwas Corynespora cassiicola ATCC 16718.

TABLE 35 Summary of TLC, HPLC/UV, and LC/MS Data for CanrenoneFermentations Evidence for 9αOH-canrenone MS: 357 HPLC-peak (M + H), TLCspot at 9αOH- 339 (−H₂O) at 9αQH- canrenone & 375 Culture AD w/UV (+NH₄)Absidia coerula ATCC n y y/n 6647 Absidia glauca ATCC n 22752Actinomucor elegans ATCC tr y tr 6476 Aspergillus flavipes tr ATCC 1030Aspergillus fumigatus tr y n ATCC 26934 Aspergillus nidulans tr y y ATCC11267 Aspergillus niger ATCC n y y 16888 Aspergillus niger ATCC n y n26693 Aspergillus ochraceus n y n ATCC 18500 Bacterium cyclo-oxydans ntr n (Searle) ATCC 12673 Beauveria bassiana ATCC tr y y 7159 Beauveriabassiana ATCC y y y 13144 Botryosphaeria obtusa y tr tr IMI 038560Calonectria decora ATCC n tr y 14767 Chaetomium cochliodes tr tr y/nATCC 10195 Comomonas testosteroni tr tr n (Searle) ATCC 11996Corynespora cassiicola y y y ATCC 16718 Cunninghamella y y y blakesleanaATCC 8688a Cunninghamella y y y echinulata ATCC 3655 Cunninghamellaelegans y y y ATCC 9245 Curcularia clavata ATCC n y y/n 22921 Curvularialunata ATCC y n n 12071 Cylindrocarpon tr n n radicicola (Searle) ATCC11011 Epicoccum humucola ATCC y y y 12722 Epicoccum oryzae ATCC tr tr tr12724 Fusarium oxysporum ATCC tr 7601 Fusarium oxysporum f. sp. n cepaeATCC 11171 Gibberella fujikuroi tr y y ATCC 14842 Gliocladiumdeliguescens y tr tr ATCC 10097 Gongronella butieri ATCC y y UV? y 22822Hypomyces chrysospermus y y y Tul. IMI 109891 Lipomyces lipofer ATCC n10792 Melanospora ornata ATCC tr n n 26180 Mortierella isabellinay y y nATCC 42613 Mucor grisco-cyanus ATCC n 1207a Mucor mucedo ATCC 4605 tr yy Mycobacterium fortuitumn NRRL B8119 Myrothecium verrucaria tr tr yATCC 9095 Nocardia aurentia n tr n (Searle) ATCC 12674 Nocardiacancicruria y y n ATCC 31548 Nocardia corallina ATCC n 19070Paecilomyces carneus n y n ATCC 46579 Penicillium chrysogenum n ATCC9480 Penicillium patulum ATCC y y y/n 24550 Penicillium purpurogenum try y ATCC 46581 Pithomyces atro- tr y tr olivaceus ATCC 6651 Pithomycescynodontis n tr tr ATCC 26150 Phycomyces blakesleeanus y y y/n IMI118496 Pycnosporium sp. ATCC y y y/n 12231 Rhizopogon sp. ATCC 36060Rhizopus arrhizus ATCC tr y n 11145 Rhizopus stolonifer ATCC n 6227bRhodococcus equi ATCC n tr n 14887 Rhodococcus equi ATCC tr tr n 21329Rhodococcus sp. ATCC n n n 19070 Rhodococcus rhodochrous n tr n ATCC19150 Saccharopolyspora y y y erythaea ATCC 11635 Sepedoniumampullosporum n n n IMI 203033 Sepedonium chrysospermum n ATCC 13378Septomyxa affinis ATCC n y UV? y/n 6737 Stachylidium bicolor y y y/nATCC 12672 Streptomyces n californicus ATCC 15436 Streptomyces ncinereocrocatus ATCC 3443 Streptomyces coelicolor n ATCC 10147Streptomyces flocculus ATCC 25453 Streptomyces fradiae n ATCC 10745Streptomyces griseus n subsp. griseus ATCC 13968 Streptomyces griseus nATCC 11984 Streptomyces hydrogenans n ATCC 19631 Streptomyces y y yhygroscopicus ATCC 27438 Streptomyces lavendulae n Panlab 105Streptomyces n paucisporogenes ATCC 25489 Streptomyces n tr trpurpurascens ATCC 25489 Streptomyces roseochromogenes ATCC 13400Streptomyces spectabilis n ATCC 27465 Stysanus microsporus ATCC 2833Syncephalastrum n racemosum ATCC 18192 Thamnidium elegans ATCC 18191Thamnostylum piriforme y tr y ATCC 8992 Thielavia terricolan n ATCC13807 Trichoderma viride ATCC n 26802 Trichothecium roseum tr y y/n ATCC12543 Verticillium theobromae y tr tr ATCC 12474

EXAMPLE 18

Various cultures were tested for effectiveness in the bioconversion ofandrostendione to 11α-hydroxyandrostendione according to the methodsgenerally described above.

A working cell bank of each of Aspergillus ochraceus NRRL 405 (ATCC18500); Aspergillus niger ATCC 11394; Aspergillus nidulans ATCC 11267;Rhizopus oryzae ATCC 11145; Rhizopus stolonifer ATCC 6227b;Trichothecium roseum ATCC 12519 and ATCC 8685 was prepared essentiallyin the manner described in Example 4. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used toinoculate transformation flasks containing the growth medium (30 ml) ofTable 15. Each culture was used for inoculation of two flasks, a totalof 16. Androstendione (300 mg) was dissolved in methanol (6 ml) at 36°C., and a 0.5 ml aliquot of this solution was introduced into each ofthe flasks. Bioconversion was carried out generally under the conditionsdescribed in Example 6 for 48 hours. After 48 hours samples of the brothwere pooled and extracted with ethyl acetate as in Example 17. The ethylacetate was concentrated by evaporation, and samples were analyzed bythin layer chromatography to determine whether a product having achromatographic mobility similar to that of 11α-hydroxy-androstendionestandard (Sigma Chemical Co., St. Louis) was present. The results areshown in Table 36. Positive results are indicated as “+”.

TABLE 36 Bioconversion of androstendione to 11 alpha-hydroxy-androstendione TLC Culture ATTC# media results Rhizopus oryzae11145 CSL + Rhizopus stolonifer 6227b CSL + Aspergillus nidulans 11267CSL + Aspergillus niger 11394 CSL + Aspergillus ochraceus NRRL 405 CSL +Aspergillus ochraceus 18500 CSL + Trichothecium roseum 12519 CSL +Trichothecium roseum  8685 CSL +

The data in Table 36 demonstrate that each of listed cultures wascapable of producing a compound from androstendione having the same Rfvalue as that of the 11α-hydroxyandrostendione standard.

Aspergillus ochraceus NRRL 405 (ATCC 18500) was retested by the sameprocedure described above, and the culture products were isolated andpurified by normal phase silica gel column chromatography using methanolas the solvent. Fractions were analyzed by thin layer chromatography.TLC plates were Whatman K6F silica gel 60 Å, 10×20 size, 250μ thickness.The solvent mixture was chloroform:methanol, 95:5, v/v. The crystallizedproduct and 11α-hydroxyandrostendione standard were both analyzed byLC-MS and NMR spectroscopy. Both compounds yielded similar profiles andmolecular weights.

EXAMPLE 19A

Various microorganisms were tested for effectiveness in thebioconversion of androstendione to 11β-hydroxyandrostendione essentiallyby the methods described above in Examples 17 and 18.

Cultures of each of Aspergillus fumigatus ATCC 26934, Aspergillus nigerATCC 16888 and ATCC 26693, Epicoccum oryzae ATCC 7156, Curvularia lunataATCC 12017, Cunninghamella blakesleeana ATCC 8688a, and Pithomycesatro-olivaceus IFO 6651 were grown essentially in the manner describedin Example 17. Growth and fermentation media (30 ml) had the compositionshown in Table 34.

The 11β-hydroxylation of androstendione by the above-listedmicroorganisms was analyzed using essentially the same methods ofproduct identification described in Examples 17 and 18. The results areset forth in Table 19A-1.

TABLE 19A-1 11β-Hydroxylation of Androstendione by VariousMicroorganisms Organism TLC LC/MS Aspergillus fumigatus + + ATCC 26934Aspergillus niger + + ATCC 16888 and ATCC 26693 Epicoccum oryzae + +ATCC 7156 Curvularia lunata + + ATCC 12017 Cunninghamellablakesleeana + + ATCC 8688a Pithomyces atro-olivaceus + + IFO 6651

In Table 19A-1, a “+” indicates a positive result, i.e., an R_(f) asexpected in thin layer chromatography and an approximately correctmolecular weight upon LC/MS.

These results demonstrate that the listed micro-organisms are capable ofcarrying out the 11β-hydroxylation of androstendione.

EXAMPLE 19B

Various microorganisms were tested for effectiveness in the conversionof mexrenone to 11β-hydroxymexrenone. Fermentation media for thisexample were prepared as described in Table 34.

The fermentation conditions and analytical methods were the same asthose in Example 17. TLC plates and the solvent system were as describedin Example 18. The rationale for chromatographic analysis is as follows:11α-hydroxymexrenone and 11α-hydroxycanrenone have the samechromatographic mobility. 11α-hydroxycanrenone and 9α-hydroxycanrenoneexhibit the same mobility pattern as 11α-hydroxyandrostendione and11β-hydroxyandrostendione. Therefore, 11β-hydroxymexrenone should havethe same mobility as 9α-hydroxycanrenone. Therefore, compounds extractedfrom the growth media were run against 9α-hydroxycanrenone as astandard. The results are shown in Table 36.

TABLE 37 Summary of TLC Data for 11β-hydroxymexrenone Formation fromMexrenone Spot Microorganism Medium¹ Character² Absidia coerula ATCC6647 M, S strong Aspergillus niger ATCC S, P faint (S) 16888 ? (P)Beauveria bassiana ATCC P strong 7159 Beauveria bassiana ATCC S, P ?, ?13144 Botryosphaeria obtusa IMI faint 038560 Cunninghamella blakesleeanaATCC 8688a S, P strong echinulata ATCC 3655 S, P strong elegans ATCC9245 S, P strong Curvularia lunata ATCC S strong 12017 Gongronellabutleri ATCC S, P strong 22822 Penicillium patulum ATCC S, P strong24550 Penicillium purpurogenum S, P strong ATCC 46581 Pithomycesatro-olivaceus S, P faint IFO 6651 Rhodococcus equi ATCC M faint 14887Saccharopolyspora erythaea M, SF faint ATCC 11635 Streptomyceshygroscopicus M, SF strong ATCC 27438 Streptomyces purpurascens M, SFfaint ATCC 25489 Thamnidium elegans ATCC S, P faint 18191 Thamnostylumpiriforme S, P faint ATCC 8992 Trichothecium roseum ATCC P, S faint (P)12543 ? (S) ¹M = Mueller-Hinton P = PYG (peptone/yeast extract/glucose)S = soybean meal SF = soybean meal plus formate ²? = questionabledifference from no substrate control

These data suggest that the majority of the organisms listed in thistable produce a product similar or identical to 11β-hydroxymexrenonefrom mexrenone.

EXAMPLE 19C

Various microorganisms were tested for effectiveness in the conversionof mexrenone to 11α-hydroxymexrenone, Δ^(1,2)-mexrenone,6β-hydroxymexrenone, 12β-hydroxymexrenone, and 9α-hydroxymexrenone.Mexrenone can be prepared in the manner set forth in Weier, U.S. Pat.No. 3,787,396 and R. M. Weier et al., J.Med.Chem., Vol. 18, pp. 817-821(1975), which are incorporated herein by reference. Fermentation mediawere prepared as described in Example 17, except that mexrenone wasincluded. The fermentation conditions were essentially the same as thosein Example 17; analytical methods were also the same as those inExamples 17 and 18. TLC plates and the solvent system were as describedin Examples 17 and 18.

The microorganisms tested and results obtained therewith are shown inTable 19C-1.

TABLE 19C-1 Production of 11α-hydroxymexrenone from Mexrenone by VariousMicroorganisms Organism TLC HPLC m/z 457:399 Beauveria bassiana + + 5:1ATCC 7159 Beauveria bassiana + + 10:1  ATCC 13144 Mortierellaisabella + + 1:1 ATCC 42613 Cunninghamella blakesleeana + + 1:1 ATCC8688a Cunninghamella echinulata + + 1:2 ATCC 3655 Cunninghamellaelegans + + 1:1 ATCC 9245 Absidia coerula + + 1:1 ATCC 6647 Aspergillusniger + + 4:1 ATCC 16888 Gongronella butieri + + 3:1 ATCC 22822Pithomyces atro-olivaceus + + 3:1 ATCC 6651 Streptomyceshygroscopicus + + 3:1 ATCC 27438

In Table 19C-1, a “+” indicates a positive result, i.e., an R_(f) asexpected in thin layer chromatography and a retention time as expectedin HPLC. m/z 417:399 indicates the peak height ratio of the 417 molecule(hydroxymexrenone) and the 399 molecule (mexrenone). The standard has a10:1 ratio of peak height for m/z 417 to m/z 399.

The product obtained from Beauveria bassiana ATCC 13144 was isolatedfrom the incubation mixture and analyzed by NMR, and the structuralprofile thereby confirmed to be 11α-hydroxymexrenone. By analogy, theproducts obtained from the other microorganisms listed in Table 19C-1were also presumed to be 11α-hydroxymexrenone.

TABLE 19C-2 Production of Δ^(1,2) -Mexrenone from Mexrenone by VariousMicroorganims Organism m/z 399 HPLC TLC Rhodococcus equi + + + ATCC148875 Bacterium cyclo-oxydans + + + ATCC 12673 Comomonastestosteroni + + + ATCC 11996 Nocardia aurentia + + + ATCC 12674Rhodococcus equi + + + ATCC 21329

In Table 19C-2, a “+” indicates a positive result, e.g., an R_(f) asexpected in thin layer chromatography, a retention time as expected inHPLC, etc.

The product obtained from Bacterium cyclo-oxydans ATCC 12673 wasisolated from the incubation mixture and analyzed by NMR, and thestructural profile thereby confirmed to be Δ^(1,2)-mexrenone. Byanalogy, the products obtained from the other microorganisms listed inTable 19C-2 were also presumed to be Δ^(1,2)-mexrenone.

Production of 6β- and 12β-hydroxymexrenone

Mortierella isabella ATCC 42613 was grown as in Example 17 in thepresence of mexrenone. The fermentation products were isolated andpurified by flash chromatography. The purified products were analyzed byLC/MS as in Examples 17 and 18, and proton NMR and carbon-13 NMR. Thedata indicated that the products included 6β- and 12β-hydroxymexrenone.

TABLE 19C-3 Production of 9α-Hydroxymexrenone from Mexrenone by VariousMicroorganisms Organism m/z 417 HPLC TLC Streptomyceshygroscopicus + + + ATCC 27438 Gongronella butleri + + + ATCC 22822Cunninghamella blakesleeana + + + ATCC 8688a Cunninghamellaechinulata + + + ATCC 3655 Cunninghamella elegans + + + ATCC 9245Mortierella isabellina + + + ATCC 42613 Absidia coerula + + + ATCC 6647Beauveria bassiana + + + ATCC 7159 Beauveria bassiana + + + ATCC 13144Aspergillus niger + + + ATCC 16888

The microorganisms listed in Table 19C-3 were grown under the sameconditions as in Example 17, in the presence of mexrenone. Thefermentation products were analyzed by TLC and LC/MS as in Examples 17and 18. A “+” indicates a positive result, e.g., an R_(f) as expected inthin layer chromatography, a retention time as expected in HPLC, etc.The data suggest that the products include 9α-hydroxymexrenone.

EXAMPLE 19D

Various microorganisms were tested for effectiveness in the conversionof canrenone to Δ^(9,11)-canrenone. The fermentation media and growthconditions were essentially the same as in Example 17, except thatcanrenone was included in the medium. The analytical methods were asdescribed in Examples 17 and 18. The microorganisms and results areshown in Table 19D-1, below.

TABLE 19D-1 Production of Δ^(9,11) -Canrenone from Canrenone by VariousMicroorganisms Organism m/z 339 HPLC TLC Bacterium cyclo-oxydans + + +ATCC 12673 Comomonas testosteroni + + + ATCC 11996 Cylindrocarponradicicola + + + ATCC 11011 Paecilomyces carneus + + + ATCC 46579Septomyxa affinis + + + ATCC 6737 Rhodococcus spp. + + + ATCC 19070

The fermentation products were analyzed by TLC and LC/MS as in Examples17 and 18. A “+” indicates a positive result, e.g., an R_(f) as expectedin thin layer chromatography, a retention time as expected in HPLC, etc.

The product obtained from Comomonas testosteroni ATCC 11996 was isolatedfrom the growth medium and analyzed by UV spectroscopy. Thespectroscopic profile confirmed the presence of Δ^(9,11)-canrenone. Byanalogy, the products obtained from the other microorganisms listed inTable 19D-1 were also presumed to be Δ^(9,11)-canrenone.

EXAMPLE 20A Scheme 1: Step 1: Method A: Preparation of5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′a,13′α-dimethyl-3′,5-dioxospirofuran-2(3H),17′α(5′H)-[7,4]metheno[4H[cyclopenta[a]phenanthrene]-5′-carbonitrile

Into a 50 gallon glass-line reactor was charged 61.2 L (57.8 kg) of DMFfollowed by 23.5 Kg of 11-hydroxycanrenone 1 with stirring. To themixture was added 7.1 kg of lithium chloride. The mixture was stirredfor 20 minutes and 16.9 kg of acetone cyanohydrin was charged followedby 5.1 kg of triethylamine. The mixture was heated to 85° C. andmaintained at this temperature for 13-18 hours. After the reaction 353 Lof water was added followed by 5.6 kg of sodium bicarbonate. The mixturewas cooled to 0° C., transferred to a 200 gallon glass-lined reactor andquenched with 130 kg of 6.7% sodium hypochlorite solution slowly. Theproduct was filtered and washed with 3×40 L portions of water to give21.4 kg of the product enamine.

H¹ NMR (DMSO-d₆): 7.6 (2H, bd), 4.53 (1H, d, J=5.9), 3.71 (1H, m),3.0-1.3 (17H, m), 1.20 (5H, m), 0.86 (3H,s), 0.51 (1H,t, J=10).

EXAMPLE 20B Preparation of7α-cyano-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-21-carboxylic acid,γ-lactone

50.0 g of 11-hydroxycanrenone and 150.0 mL of dimethylacetamide wereadded to a clean, dry three-necked flask equipped with a mechanicalstirrer, condenser, thermocouple and heating mantle. 16.0 mL of asulfuric acid solution (prepared by mixing 50.0 mL of sulfuric acid(98.7% Baker grade) with 50.0 mL of water) was added to this mixture. Asodium cyanide solution comprising 15.6 g of sodium cyanide and 27.0 mLof water was then added.

The resulting mixture was heated at 80° C. for 7 hours, the degree ofconversion being periodically checked by TLC or HPLC. Afterapproximately 7 hours, HPLC of the mixture indicated the presence of the7-cyano compound. The mixture was then stirred overnight and allowed tocool to room temperature (about 22° C.). 200 mL of water was added tothe mixture followed by 200 mL of methylene chloride and the resultingtwo phase mixture stirred and the phases were then allowed to separate.The aqueous layer was a gel. 100 mL of sodium bicarbonate solution wasadded to the aqueous layer in an unsuccessful attempt to break up thegel. The aqueous layer was then discarded.

The separated methylene chloride layer was washed with 100 mL of waterand the resulting two phase mixture stirred. The phases were thenallowed to separate and the separated methylene chloride layer wasfiltered through 200 g of silica gel (Aldrich 200-400 mesh, 60 Å). Thefiltrate was concentrated to dryness under reduced pressure at 45° C.using a water aspirator to provide about 53.9 g of a crude solidproduct. The crude solid product then was dissolved in 50 mL ofmethylene chloride and treated with 40 mL of 4N hydrochloric acid in aseparatory funnel and the two phase mixture allowed to separate. Themethylene chloride layer was washed with 50 mL of water. The combinedaqueous layers were extracted with 50 mL of methylene chloride chloride.The combined methylene chloride layers were then dried over sodiumsulfate to provide 45 g of a solid which was a mixture of11α-hydroxycanrenone and the product,7α-cyano-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-21-carboxylic acid,γ-lactone.

A sample of the product was analyzed by HPLC (column: 25 cm×4.6 mm, 5μAltima C₁₈LL); solvent gradient: solvent A=water/trifluoroaceticacid=99.9/0.1, solvent B=acetonitrile/trifluoroacetic acid=99.9/0.1,flow rate=1.00 mL/minute, gradient=65:30 (v/v) (A:B—initial), 35:65(v/v) (A:B—after 20 minutes), 10:90 (v/v) (A:B—after 25 minutes); diodearray detector) which revealed a λ_(max) of 238 nm.

The reaction mixture was analyzed by HPLC-NMR using the followingconditions: HPLC—column: Zorbax RX-C8 (25 cm×4.6 mm, 5μ) using a solventgradient from 75% D₂O, 25% acetonitrile to 25% D2O, 75% acetonitrileover 25 minutes with a flow of 1 mL/minute; ¹H NMR (obtained using WETsolvent suppression): 5.84 (s,1H), 4.01 (m,1H), 3.2 (m,1H), 2.9-1.4 (m,integral not meaningful due to solvent suppression of acetonitrile),0.93-0.86 (s, overlapping 3H, and t,2H).

EXAMPLE 20C Preparation of5β,7α-dicyano-17-hydroxy-3-oxo-17α-pregnane-21-carboxylic acid,γ-lactone

102 g (0.3 mol) of 17-hydroxy-3-oxo-17α-pregna-4,6-diene-21-carboxylicacid, γ-lactone (canrenone) was slurried with 46.8 g (0.72 mol) ofpotassium cyanide, 78.6 mL (1.356 mols) of acetic acid, and 600 mL ofmethanol in a three liter, three neck, round bottom flask. 64.8 mL (0.78mol) of pyrrolidine was added to the mixture and the combined slurryheated to reflux (64° C.) and maintained for about 1.5 hours. Thetemperature of the slurry was then lowered to 25° C. to 30° C. over aten minute period with a cooling bath. 120 mL of a concentratedhydrochloric acid was slowly added during the cooldown as a tan coloredsolid precipitated.

The mixture was stirred at 25° C. to 30° C. for 1.5 hours, then anadditional 500 mL of water added in 30 minutes. The mixture was cooledto 5° C. with an ice bath and the pH adjusted from 3 to 5.5 (monitoredusing pH strips) with the addition of 100 mL of aqueous 9.5M sodiumhydroxide (0.95 mol). Excess cyanide was destroyed with the addition ofhousehold bleach. 25 mL (0.020 mol) was added to achieve a negativestarch iodide test. The cold mixture (10° C.) was filtered and the solidwashed with water until the rinse exhibited a neutral pH (pH strips).The solid was dried at 60° C. to a constant weight of 111.4 g.

The isolated solid melted at 244° C. to 246° C. on a Fisher Johns block.A methanol solution containing the solid exhibited no absorptionthroughout the UV region of 210 to 240 nm. IR (CHCl₃)cm⁻¹2222 (cyanide),1775 (lactone), 1732 (3-keto). ¹H NMR (pyridine d₅) ppm 0.94 (s,3H),1.23 (s,3H).

EXAMPLE 21A Scheme 1: Step 2: Preparation of4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile

Into a 200 gallon glass-lined reactor was charged 50 kg of enamine 2,approximately 445 L of 0.8 N dilute hydrochloric acid and 75 L ofmethanol. The mixture was heated to 80° C. for 5 hours, cooled to 0° C.for 2 hours. The solid product was filtered to give 36.5 kg of dryproduct diketone.

H¹ NMR (DMSO-d₆) 4.53(1H, d, J=6) 3.74 (2H, m), 2.73 (1H,dd, J=14,7)2.65-2.14 (8H, m), 2.05 (1H, t, J=11), 1.98-1.71 (4H,m), 1.64 (1H, m),1.55 (1H, dd, J=13, 5), 1.45-1.20 (7H, m), 0.86 (3H,s).

EXAMPLE 21B Scheme 1: Steps 1 and 2: In Situ Preparation of4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano-[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrilefrom 11α-hydroxycanrenone

Into a reactor fitted with a cooling condenser, mechanical stirrer,heating mantle and controller, and funnel was charged 100 g (280.54mmol) of 11-hydroxycanrenone prepared prepared as in the manner ofExample 1 followed by 300 mL of dimethylacetamide (Aldrich). The mixturewas stirred until the 11-hydroxycanrenone dissolved. To this mixture wasadded 31.5 mL of 50% sulfuric acid (Fisher) which caused the temperatureof the resulting mixture to rise about 10° C. to 15° C. A sodium cyanidesolution prepared by dissolving 31.18 g (617.20 mmol) (Aldrich) ofsodium cyanide in 54 mL of deionized water was then added to the11α-hydroxycanrenone mixture over a 2 to 3 minute period. Thetemperature of the resulting mixture rose about 20° C. to 25° C. afteraddition of the sodium cyanide solution.

The mixture was heated to 80° C. and maintained at this temperature for2-3 hours. Once HPLC analysis indicated the reaction for the conversionof the 11α-hydroxycanrenone to the enamine was substantially complete(greater than 98% conversion), the heat source was removed. Withoutisolation of the enamine contained in the mixture, an additional 148 mLof 50% sulfuric acid was added to the mixture over a 3-5 minute period.Over a 10 minute period 497 mL of deionized water was then added to themixture.

The mixture was heated to 102° C. and maintained at that temperatureuntil approximately 500 g of distillate had been removed from themixture. During the reaction/distillation, 500 mL of deionized water wasadded to the mixture in four separate 125 mL portions. Each portion wasadded to the mixture after an equivalent amount of distillate(approximately 125 mL) had been removed. The reaction continued for over2 hours. When HPLC analysis indicated that the reaction hydrolyzing theenamine to the diketone was substantially completed (greater than 98%conversion), the mixture was cooled to about 80° C. over a 20 minuteperiod.

The mixture was filtered through a glass funnel. The reactor was rinsedwith 1.2 L of deionized water to remove residual product. The solid onthe filter was washed three times using approximately equal portions(about 0.4 L) of the rinse water. A 1 L solution of methanol anddeionized water (1:1 v/v) was prepared in the reactor and the filtratewas washed with 500 mL of this solution. The filtrate was then washed asecond time with the remaining 500 mL of the methanol/water solution.Vacuum was applied to the funnel to dry the filtrate sufficiently fortransfer. The filtrate was transferred to a drying oven where it wasdried under vacuum for 16 hours to yield 84 g of dry product diketone,4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano-[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile.HPLC assay indicated 94% of the desired diketone.

EXAMPLE 22 Scheme 1: Step 3A: Method A: Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

A 4-neck 5-L bottom flask was equipped with mechanical stirrer, pressureequalizing addition funnel with nitrogen inlet tube, thermometer andcondenser with bubbler. The bubbler was connected via tygon tubing totwo 2-L traps, the first of which was empty and placed to preventback-suction of the material in the second trap (1 L of concentratedsodium hypochlorite solution) into the reaction vessel. The diketone 3(79.50 g; [weight not corrected for purity, which was 85%]) was added tothe flask in 3 L methanol. A 25% methanolic sodium methoxide solution(64.83 g) was placed in the funnel and added dropwise, with stirringunder nitrogen, over a 10 minute period. After the addition wascomplete, the orangish yellow reaction mixture was heated to reflux for20 hours. After this period, 167 mL of 4 N HCl was added (Caution: HCNevolution at this point!) dropwise through the addition funnel to thestill refluxing reaction mixture. The reaction mixture lightened incolor to a pale golden orange. The condenser was then replaced with atake-off head and 1.5 L of methanol was removed by distillation while1.5 L of water was simultaneously added to the flask through the funnel,in concert with the distillation rate. The reaction mixture was cooledto ambient temperature and extracted twice with 2.25 L aliquots ofmethylene chloride. The combined extracts were washed successively with750 mL aliquots of cold saturated NaCl solution, 1N NaOH and again withsaturated NaCl. The organic layer was dried over sodium sulfateovernight, filtered and reduced in volume to ˜250 mL in vacuo. Toluene(300 mL) was added and the remaining methylene chloride was strippedunder reduced pressure, during which time the product began to form onthe walls of the flask as a white solid. The contents of the flask werecooled overnight and the solid was removed by filtration. It was washedwith 250 mL toluene and twice with 250 mL aliquots of ether and dried ona vacuum funnel to give 58.49 g of white solid was 97.3% pure by HPLC.On concentrating the mother liquor, an additional 6.76 g of 77.1% pureproduct was obtained. The total yield, adjusted for purity, was 78%.

H¹ NMR (CDCl₃): 5.70 (1H,s), 4.08 (1H,s), 3.67 (3H,s), 2.9-1.6 (19H, m),1.5-1.2 (5H, m), 1.03 (3H.s).

EXAMPLE 23 Scheme 1: Step 3B: Conversion of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone toMethyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone

A 5-L four neck flask was equipped as in the above example, except thatno trapping system was installed beyond the bubbler. A quantity of138.70 g of the hydroxyester was added to the flask, followed by 1425 mLmethylene chloride, with stirring under nitrogen. The reaction mixturewas cooled to −5° C. using a salt/ice bath. Methanesulfonyl chloride(51.15 g, 0.447 mole) was added rapidly, followed by the slow dropwiseaddition of triethylamine (54.37 g) in 225 mL methylene chloride.Addition, which required ˜30 minutes, was adjusted so that thetemperature of the reaction never rose about 5° C. Stirring wascontinued for 1 hour post-addition, and the reaction contents weretransferred to a 12-L separatory funnel, to which was added 2100 mLmethylene chloride. The solution was washed successively with 700 mLaliquots each of cold 1N HCl, 1N NaOH, and saturated aqueous NaClsolution. The aqueous washes were combined and back-extracted with 3500mL methylene chloride. All of the organic washes were combined in a 9-Ljug, to which was added 500 g neutral alumina, activity grade II, and500 g anhydrous sodium sulfate. The contents of the jug were mixed wellfor 30 minutes and filtered. The filtrate was taken to dryness in vacuoto give a gummy yellow foam. This was dissolved in 350 mL methylenechloride and 1800 mL ether was added dropwise with stirring. The rate ofaddition was adjusted so that about one-half of the ether was added over30 minutes. After about 750 mL had been added, the product began toseparate as a crystalline solid. The remaining ether was added in 10minutes. The solid was removed by filtration, and the filter cake waswashed with 2 L of ether and dried in a vacuum oven at 50° C. overnight,to give 144.61 g (88%) nearly white solid, m.p. 149°-150° C. Materialprepared in this fashion is typically 98-99% pure by HPLC (area %). Inone run, material having a melting point of 153°-153.5° C. was obtained,with a purity, as determined by HPLC area, of 99.5%.

H¹ NMR (CDCl₃): 5.76 (1H,s), 5.18 (1H,dt), 3.68(3H,s), 3.06 (3H,s), 2.85(1H,m), 2.75-1.6 (19H, m), 1.43 (3H,s), 1.07 (3H,s).

EXAMPLE 24 Scheme 1: Step 3C: Method A: Preparation of 7-Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone

A 1-L four neck flask was equipped as in the second example. Formic acid(250 mL) and acetic anhydride (62 mL) were added to the flask withstirring under nitrogen. Potassium formate (6.17 g) was added and thereaction mixture was heated with an oil bath to an internal temperatureof 40° C. (this was later repeated at 70° C. with better results) for 16hours. After 16 hours, the mesylate was added and the internaltemperature was increased to 100° C. Heating and stirring were continuedfor 2 hours, after which the solvent was removed in vacuo on a rotavap.The residue was stirred with 500 mL ice water for fifteen minutes, thenextracted twice with 500 mL aliquots of ethyl acetate. The organicphases were combined and washed successively with cold 250 mL aliquotsof saturated sodium chloride solution (two times), 1 N sodium hydroxidesolution, and again with saturated sodium chloride. The organic phasewas then dried over sodium sulfate, filtered and taken to dryness invacuo to give a yellowish white foam, which pulverized to a glass whentouched with a spatula. The powder that formed, 14.65 g analyzed (byHPLC area %) as a mixture of 82.1% 7-Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone;7.4% 7-Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,11-diene-7α,21-dicarboxylate, γ-Lactone; and5.7% 9α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,bis(γ-lactone).

H¹ NMR (CDCl₃): 5.74 (1H, s), 5.67 (1H,m), 3.61 (3H,s), 3.00 (1H,m),2.84 (1H, ddd, J=2,6,15), 2.65-2.42 (6H,m), 2.3-2.12 (5H,m), 2.05-1.72(4H,m), 1.55-1.45 (2H,m), 1.42 (3H,s), 0.97 (3H,s).

EXAMPLE 25 Scheme 1: Step 3C: Method B: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone

A 5-L four neck flask was equipped as in the above example and 228.26 gacetic acid and 41.37 g sodium acetate were added with stirring undernitrogen. Using an oil bath, the mixture was heated to an internaltemperature of 100° C. The mesylate (123.65 g) was added, and heatingwas continued for thirty minutes. At the end of this period, heating wasstopped and 200 mL of ice water was added. The temperature dropped to40° C. and stirring was continued for 1 hour, after which the reactionmixture was poured slowly into 1.5 L of cold water in a 5-L stirredflask. The product separated as a gummy oil. The oil was dissolved in 1L ethyl acetate and washed with 1 L each cold saturated sodium chloridesolution, 1 N sodium hydroxide, and finally saturated sodium chlorideagain. The organic phase was dried over sodium sulfate and filtered. Thefiltrate was taken to dryness in vacuo to give a foam which collapsed toa gummy oil. This was triturated with ether for some time and eventuallysolidified. The solid was filtered and washed with more ether to afford79.59 g of a yellow white solid. This consisted of 70.4% of the desiredΔ^(9,11) enester 6, 12.3% of the Δ^(11,12) enester 8, 10.8% of the7-α,9-α-lactone 9 and 5.7% unreacted 5.

EXAMPLE 26 Scheme 1: Step 3D: Method A: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

A 4-neck jacketed 500 mL reactor was equipped with mechanical stirrer,condenser/bubbler, thermometer and addition funnel with nitrogen inlettube. The reactor was charged with 8.32 g of the crude enester in 83 mLmethylene chloride, with stirring under nitrogen. To this was added 4.02g dibasic potassium phosphate, followed by 12 mL oftrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 8° C. To theaddition funnel 36 mL of 30% hydrogen peroxide was added over a 10minute period. The initially pale yellow colored reaction mixture turnedalmost colorless after the addition was complete. The reaction mixtureremained at 9±1° C. throughout the addition and on continued stirringovernight (23 hours total) Methylene chloride (150 mL) was added to thereaction mixture and the entire contents were added to ˜250 mL icewater. This was extracted three times with 150 mL aliquots of methylenechloride. The combined methylene chloride extracts were washed with 400mL cold 3% sodium sulfite solution to decompose any residual peroxide.This was followed by a 330 mL cold 1 N sodium hydroxide wash, a 400 mLcold 1 N hydrochloric acid wash, and finally a wash with 400 mL brine.The organic phase was dried over magnesium sulfate, filtered, and thefilter cake was washed with 80 mL methylene chloride. Solvent wasremoved in vacuo to give 9.10 g crude product as a pale yellow solid.This was recrystallized from ˜25 mL 2-butanone to give 5.52 g nearlywhite crystals. A final recrystallization from acetone (˜50 mL gave 3.16g long, acicular crystals, mp 241-243° C.

H¹ NMR (CDCl₃): 5.92 (1H,s), 3.67(3H,s), 3.13 (1H,d,J=5), 2.89 (1H, m),2.81-2.69 (15H,m), 1.72 (1H, dd, J=5,15), 1.52-1.22 (5H,m), 1.04 (3H,s).

EXAMPLE 27 Scheme 1: Step 3: Option 1: From4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Diketone (20 g) was charged into a clean and dried reactor followed bythe addition of 820 ml of MeOH and 17.6 ml of 25% NaOMe/MeOH solution.The reaction mixture was heated to reflux condition (˜67° C.) for 16-20hours. The product was quenched with 40 mL of 4N HCl. The solvent wasremoved at atmospheric pressure by distillation. 100 mL of toluene wasadded and the residual methanol was removed by azeotrope distillationwith toluene. After concentration, the crude hydroxyester 4 wasdissolved in 206 mL of methylene chloride and cooled to 0° C.Methanesulfonyl chloride (5 mL) was added followed by a slow addition of10.8 ml of triethylamine. The product was stirred for 45 minutes. Thesolvent was removed by vacuum distillation to give the crude mesylate 5.

In a separate dried reactor was added 5.93 g of potassium formate, 240mL of formic acid and followed by 118 mL of acetic anhydride. Themixture was heated to 70° C. for 4 hours.

The formic acid mixture was added to the concentrated mesylate solution5 prepared above. The mixture was heated to 95-105° C. for 2 hours. Theproduct mixture was cooled to 50° C. and the volatile components wereremoved by vacuum distillations at 50° C. The product was partitionedbetween 275 ml of ethyl acetate and 275 ml of water. The aqueous layerwas back extracted with 137 ml of ethyl acetate, washed with 240 ml ofcold 1N sodium hydroxide solution and then 120 ml of saturated NaCl.After phase separation, the organic layer was concentrated to undervacuum distillation to give crude enester.

The product was dissolved in 180 mL of methylene chloride and cooled to0 to 15° C. 8.68 g of dipotassium hydrogen phosphate was added followedby 2.9 mL of trichloroacetonitrile. A 78 mL solution of 30% hydrogenperoxide was added to the mixture over a 3 minute period. The reactionmixture was stirred at 0-15° C. for 6-24 hours. After the reaction, thetwo phase mixture was separated. The organic layer was washed with 126mL of 3% sodium sulfite solution, 126 mL of 0.5 N sodium hydroxidesolution, 126 mL of 1 N hydrochloric acid and 126 mL of 10% brine. Theproduct was dried over anhydrous magnesium sulfate or filtered overCelite and the solvent methylene chloride was removed by distillation atatmospheric pressure. The product was crystallized from methylethylketone twice to give 7.2 g of epoxymexrenone.

EXAMPLE 28 Scheme 1: Step 3: Option 2: Conversion of1′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactonewithout intermediate isolation

A 4-neck 5-L round bottom flask was equipped with mechanical stirrer,addition funnel with nitrogen inlet tube, thermometer and condenser withbubbler attached to a sodium hypochlorite scrubber. The diketone (83.20g) was added to the flask in 3.05 L methanol. The addition funnel wascharged with 67.85 g of a 25% (w:w) solution of sodium methoxide inmethanol. With stirring under nitrogen, the methoxide was added dropwiseto the flask over a 15 minute period. A dark orange/yellow slurrydeveloped. The reaction mixture was heated to reflux for 20 hours and175 mL 4 N hydrochloric acid was added dropwise while refluxingcontinued. (Caution, HCN evolution during this operation!) The refluxcondenser was replaced with a takeoff head and 1.6 L of methanol wasremoved by distillation while 1.6 L of aqueous 10% sodium chloridesolution was added dropwise through the funnel, at a rate to match thedistillation rate. The reaction mixture was cooled to ambienttemperature and extracted twice with 2.25 L of aliquots of methylenechloride. The combined extracts were washed with cold 750 mL aliquots of1 N sodium hydroxide and saturated sodium chloride solution. The organiclayer was dried by azeotropic distillation of the methanol at oneatmosphere, to a final volume of 1 L (0.5% of the total was removed foranalysis).

The concentrated organic solution (hydroxyester) was added back to theoriginal reaction flask equipped as before, but without the HCN trap.The flask was cooled to 0° C. and 30.7 g methanesulfonyl chloride wasadded with stirring under nitrogen. The addition funnel was charged with32.65 g triethylamine, which was added dropwise over a 15 minute period,keeping the temperature at 5° C. Stirring was continued for 2 hours,while the reaction mixture warmed to ambient. A column consisting of 250g Dowex 50 W×8-100 acid ion exchange resin was prepared and was washedbefore using with 250 mL water, 250 mL methanol and 500 mL methylenechloride. The reaction mixture was run down this column and collected. Afresh column was prepared and the above process was repeated. A third250 g column, consisting of Dowex 1×8-200 basic ion exchange resin wasprepared and pretreated as in the acid resin treatment described above.The reaction mixture was run down this column and collected A fourthcolumn of the basic resin was prepared and the reaction mixture againwas run down the column and collected. Each column pass was followed bytwo 250 mL methylene chloride washes down the column, and each passrequired ˜10 minutes. The solvent washes were combined with the reactionmixture and the volume was reduced in vacuo to ˜500 mL and 2% of thiswas removed for qc. The remainder was further reduced to a final volumeof 150 mL (crude mesylate solution).

To the original 5-L reaction set-up was added 960 mL formic acid, 472 mLacetic anhydride and 23.70 g potassium formate. This mixture was heatedwith stirring under nitrogen to 70° C. for 16 hours. The temperature wasthen increased to 100° C. and the crude mesylate solution was added overa thirty minute period via the addition funnel. The temperature droppedto 85° C. as methylene chloride was distilling out of the reactionmixture. After all of it had been removed, the temperature climbed backto 100° C., and was held there for 2.5 hours. The reaction mixture wascooled to 40° C. and the formic acid was removed under pressure untilthe minimum stir volume had been reached (˜150 mL). The residue wascooled to ambient and 375 mL methylene chloride was added. The dilutedresidue was washed with cold 1 L portions of saturated sodium chloridesolution, 1 N sodium carbonate, and again with sodium chloride solution.The organic phase was dried over magnesium sulfate (150 g), and filteredto give a dark reddish brown solution (crude enester solution).

A 4-neck jacketed 1 L reactor was equipped with mechanical stirrer,condenser/bubbler, thermometer and addition funnel with nitrogen inlettube. The reactor was charged with the crude enester solution (estimated60 g) in 600 mL methylene chloride, with stirring under nitrogen. Tothis was added 24.0 g dibasic potassium phosphate, followed by 87 mLtrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 10° C. To theaddition funnel 147 mL 30% hydrogen peroxide was added mixture over a 30minute period. The initially dark reddish brown colored reaction mixtureturned a pale yellow after the addition was complete. The reactionmixture remained at 10±1° C. throughout the addition and on continuedstirring overnight (23 hours total). The phases were separated and theaqueous portion was extracted twice with 120 mL portions of methylenechloride. The combined organic phases were then washed with 210 mL 3%sodium sulfite solution was added. This was repeated a second time,after which both the organic and aqueous parts were negative forperoxide by starch/iodide test paper. The organic phase was successivelywashed with 210 mL aliquots of cold 1 N sodium hydroxide, 1 Nhydrochloric acid, and finally two washes with brine. The organic phasewas dried azeotropically to a volume of ˜100 mL, fresh solvent was added(250 mL and distilled azeotropically to the same 100 mL and theremaining solvent was removed in vacuo to give 57.05 g crude product asa gummy yellow foam. A portion (51.01 g) was further dried to a constantweight of 44.3 g and quantitatively analyzed by HPLC. It assayed at27.1% epoxymexrenone.

EXAMPLE 29 Formation of 3-ethoxy-11α-hydroxy-androsta-3,5-diene-17-onefrom 11α-hydroxyandrostendione

11α-Hydroxyandrostendione (429.5 g) and toluene sulfonic acid hydrate(7.1) were charged to a reaction flask under nitrogen. Ethanol (2.58 L)was added to the reactor, and the resulting solution cooled to 5° C.Triethyl orthoformate (334.5 g) was added to the solution over a 15minute period at 0° to 15° C. After the triethyl orthoformate additionwas complete the reaction mixture was warmed to 40° C. and reacted atthat temperature for 2 hours, after which the temperature was increasedto reflux and reaction continued under reflux for an additional 3 hours.The reaction mixture was cooled under vacuum and the solvent removedunder vacuum to yield 3-ethoxy-11α-hydroxy-androsta-3,5-diene-17-one.

EXAMPLE 30 Formation of Enamine from 11α-hydroxycanrenone Scheme 1: Step3A: Method B: Preparation of5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′a,13′α-dimethyl-3′,5dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H[cyclopenta[a]phenanthrene[-5′-carbonitrile.

Sodium cyanide (1.72 g) was placed in 25 mL 3-neck flask fitted with amechanical stirrer. Water (2.1 mL) was added and the mixture was stirredwith heating until the solids dissolved. Dimethylformamide (15 mL) wasadded followed by 11α-hydroxycanrenone (5.0 g). A mixture of water (0.4mL) and sulfuric acid (1.49 g) was added to mixture. The mixture washeated to 85° C. for 2.5 hours at which time HPLC analysis showedcomplete conversion to product. The reaction mixture was cooled to roomtemperature. Sulfuric acid (0.83 g) was added and the mixture stirredfor one half hour. The reaction mixture was added to 60 mL water cooledin an ice bath. The flask was washed with 3 mL DMF and 5 mL water. Theslurry was stirred for 40 min. and filtered. The filter cake was washedtwice with 40 mL water and dried in a vacuum oven at 60° C. overnight toyield the 11α-hydroxy enamine, i.e.,5′R(5′α),7′β-20′-aminohexadecahydro-11′β-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile (4.9 g).

EXAMPLE 31 One-pot Conversion of 11α-hydroxycanrenone to Diketone

Sodium cyanide (1.03 g) was added to a 50 mL 3-neck flask fitted with amechanical stirrer. Water (1.26 mL) was added and the flask was heatedslightly to dissolve the solid. Dimethylacetamide [or dimethyformamide](9 mL) was added followed by 11α-hydroxycanrenone (3.0 g). A mixture ofsulfuric acid (0.47 mL) and water (0.25 mL) was added to the reactionflask while stirring. The mixture was heated to 95° C. for 2 hours. HPLCanalysis indicated that the reaction was complete. Sulfuric acid (0.27mL) was added and the mixture stirred for 30 min. Additional water (25mL) and sulfuric acid (0.90 mL) were introduced and the reaction mixturestirred for 16 hours. The mixture was then cooled in an ice bath to5-10° C. The solid was isolated by filtering through a sintered glassfilter followed by washing twice with water (20 mL). The solid diketone,i.e.,4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclo-penta[a]phenanthrene]-5′β(2′H)-carbonitrilewas dried in a vacuum oven to yield 3.0 g of a solid.

EXAMPLE 32A-1 Scheme 1: Step 3A: Method B: Preparation of MethylHydrogen 11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone

A suspension of 5.0 g of the diketone produced in the manner describedin Example 31 in methanol (100 mL) was heated to reflux and a 25%solution of potassium methoxide in methanol (5.8 mL) was added over 1min. The mixture became homogeneous. After 15 min., a precipitate waspresent. The mixture was heated at reflux and again became homogeneousafter about 4 hours. Heating at reflux was continued for a total of 23.5hours and 4.0 N HCl (10 mL) was added. A total of 60 mL of a solution ofhydrogen cyanide in methanol was removed by distillation. Water (57 mL)was added to the distillation residue over 15 min. The temperature ofthe solution was raised to 81.5° C. during water addition and anadditional 4 mL of hydrogen cyanide/methanol solution was removed bydistillation. After water addition was complete, the mixture becamecloudy and the heat source was removed. The mixture was stirred for 3.5hours and product slowly crystallized. The suspension was filtered andthe collected solid was washed with water, dried in a stream of air onthe funnel, and dried at 92° (26 in. Hg) for 16 hours to give 2.98 g ofan off-white solid. The solid was 91.4% of the hydroxyester, i.e.,methyl hydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone by weight. The yield was 56.1%.

EXAMPLE 32A-2 Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

The diketone (40 g) produced in the manner described in Example 31 ischarged to a clean, dry, jacketed 1 L reactor equipped with a bottomdrain, condenser, RTD probe and fraction collecting receiver. Methanol(800 mL) is then charged to the reactor and the mixture stirred. Theresulting slurry is heated to about 60° C. to 65° C. and a 25% solutionof potassium methoxide (27.8 mL) is added. The mixture becomeshomogeneous.

The mixture is heated at reflux. After about 1.5 hours at reflux anadditional 16.7 mL of 25% potassium methoxide solution is added to themixture while maintaining reflux. The mixture is maintained at refluxfor an additional 6 hours. Conversion of diketone to hydroxyester isanalyzed by HPLC. Once HPLC indicates the ratio of diketone tohydroxyester is less than about 10%, a charge of 77 mL of 4M HCl (acomparable amount of 1.5 M to 3 M sulfuric acid could be substituted forthe hydrochloric acid) is added to the mixture over a period of about 15minutes as refluxing is continued.

The mixture is then distilled and about 520 mL of methanol/HCNdistillate is collected and discarded. The concentrated mixture iscooled to about 65° C. About 520 mL of water is added to the mixtureover a period of about 90 minutes and the temperature is maintained atabout 65° C. during the addition. The mixture is gradually cooled toabout 15° C. over a period of about four hours, and then is stirred andmaintained at about 15° C. for an additional two hours. The mixture isfiltered and the filtered product is washed twice with about 200 mL ofwater each time. The filtered product is dried in vacuo (90° C., 25 mmHg). Approximately 25 to 27 g of an off-white solid comprisingprincipally methyl hydrogen11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone isobtained.

EXAMPLE 32B-1 Preparation of 7-methyl hydrogen5β-cyano-11α,17-dihydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone

A reaction flask was charged with 4.1 g of the diketone produced in themanner described in Example 31, 75 mL of methanol and 1 mL of 1Nmethanolic sodium hydroxide. The suspension was stirred at roomtemperature. A homogeneous solution was obtained within minutes and aprecipitate observed after about 20 minutes. Stirring was continued for70 minutes at room temperature. At the end of this time the solid wasfiltered and washed with methanol. The solid was dried in a steamcabinet resulting in 3.6 g of 7-methyl hydrogen5β-cyano-11α,17-dihydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone.

1H NMR (CDCl₃) ppm 0.95 (s,3H), 1.4 (s,3H), 3.03 (d,1H,J15), 3.69(s,3H), 4.1 (m,1H).

13C NMR (CDCl₃) ppm 14.6, 19.8, 22.6, 29.0, 31.0, 33.9, 35.17, 35.20,36.3, 37.7, 38.0, 38.9, 40.8, 42.8, 43.1, 45.3, 45.7, 47.5, 52.0, 68.0,95.0, 121.6, 174.5, 176.4, 207.0.

EXAMPLE 32B-2 Preparation of 7-methyl hydrogen5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone

To 2.0 g (4.88 mmol) of the 9,11-epoxydiketone of Formula 21 suspendedin 30 mL of anhydrous methanol was added 0.34 mL (2.4 mmol) oftriethylamine. The suspension was heated to reflux and after 4.5 hoursno starting material remained as judged by HPLC (Zorbax SB-C8 150×4.6mm, 2 ml/min., linear gradient 35:65 A:B to 45:55 A:B over 15 min,A=acetonitrile/methanol 1:1, B=water/0.1% trifluoroacetic acid,detection at 210 nm). The mixture was allowed to cool and maintained atabout 25 degrees for about 16 hours. The resulting suspension wasfiltered to give 1.3 g of 7-methyl hydrogen5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone as a white solid. The filtrate was concentrated to dryness ona rotary evaporator and the residue was triturated with 3-5 mL ofmethanol. Filtration gave an additional 260 mg of 7-methyl hydrogen5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone. The yield was 74.3%.

1H-nmr (400 MHz, deuterochloroform) d 1.00 (s,3H), 1.45 (m, 1H), 1.50(s,3H), 1.65 (m, 2H), 2.10 (m, 2H), 2.15-2.65 (m, 8H), 2.80 (m, 1H),2.96 (m, 1H), 3.12 (d, J=13, 1H), 3.35, (d, J=7, 1H), 3.67 (s, 3H).

EXAMPLE 32C Preparation of5β-cyano-11-α,17-dihydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylic acid,γ-lactone

A reaction flask was charged with 6.8 g of the diketone (prepared in themanner described in Example 31), 68 mL of acetonitrile, 6.0 g of sodiumacetate and 60 mL of water. The mixture was warmed and stirred atreflux. After about 1.5 hours the mixture was almost homogeneous. At theend of three hours 100 mL of water was added as 50 mL of acetonitrilewas distilled. The mixture was cooled and the precipitated solid (1.7 g)was removed via filtration. The filtrate (pH=5.5) was treated withhydrochloric acid to reduce the pH to about 4.5 and a solidprecipitated. The solid was isolated, washed with water and dried togive 4.5 g of5β-cyano-11-α,17-dihydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylic acid,γ-lactone.

¹H NMR (DMSO) ppm 0.8 (s,3H), 1.28 (s,3H), 3.82 (m,1H).

¹³C NMR (DMSO) ppm 14.5, 19.5, 22.0, 28.6, 30.2, 33.0, 34.1, 34.4, 36.0,37.5, 37.7, 38.5, 42.4, 42.6, 45.08, 45.14, 47.6, 94.6, 122.3, 176.08,176.24, 207.5.

EXAMPLE 33 Scheme 1: Step 3A: Method C: Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Diketone (30 g) prepared in the manner described in Example 31 wascharged into a cleaned and dried 3-neck reaction flask equipped with athermometer, a Dean Stark trap and a mechanical stirrer. Methanol (24mL) was charged to the reactor at room temperature (22° C.) and theresulting slurry stirred for 5 min. A 25% by weight solution of sodiummethoxide in methanol (52.8 mL) was charged to the reactor and themixture stirred for 10 min. at room temperature during which thereaction mixture turned to a light brown clear solution and a slightexotherm was observed (2-3° C.). The addition rate was controlled toprevent the pot temperature from exceeding 30° C. The mixture wasthereafter heated to reflux conditions (about 67° C.) and continuedunder reflux for 16 hrs. A sample was then taken and analyzed by HPLCfor conversion. The reaction was continued under reflux until theresidual diketone was not greater than 3% of the diketone charge. Duringreflux 4 N HCl (120 mL) was charged to the reaction pot resulting in thegeneration of HCN which was quenched in a scrubber.

After conclusion of the reaction, 90-95% of the methanol solvent wasdistilled out of the reaction mixture at atmospheric pressure. Headtemperature during distillation varied from 67-75° C. and the distillatewhich contained HCN was treated with caustic and bleach before disposal.After removal of methanol the reaction mixture was cooled to roomtemperature, solid product beginning to precipitate as the mixturecooled in the 40-45° C. range. An aqueous solution containing optionally5% by weight sodium bicarbonate (1200 mL) at 25° C. was charged to thecooled slurry and the resultant mixture then cooled to 0° C. in about 1hr. Sodium bicarbonate treatment was effective to eliminate residualunreacted diketone from the reaction mixture. The slurry was stirred at0° C. for 2 hrs. to complete the precipitation and crystallization afterwhich the solid product, Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone, wasrecovered by filtration and the filter cake washed with water (100 mL).The product was dried at 80-90° C. under 26″ mercury vacuum to constantweight. Water content after drying was less than 0.25% by weight.Adjusted molar yield was around 77-80% by weight.

EXAMPLE 34 Scheme 1: Step 3A: Method D: Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Diketone as prepared in accordance with Example 31 (1 eq.) was reactedwith sodium methoxide (4.8 eqs.) in a methanol solvent in the presenceof zinc iodide (1 eq.). Work up of the reaction product can be either inaccordance with the extractive process described herein, or by anon-extractive process in which methylene chloride extractions, brineand caustic washes, and sodium sulfate drying steps are eliminated. Alsoin the non-extractive process, toluene was replaced with 5% by weightsodium bicarbonate solution. Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone wasisolated as the product.

EXAMPLE 35 Scheme 1: Step 3C: Method C: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone

The hydroxyester prepared as by Example 34 (1.97 g) was combined withtetrahydrofuran (20 mL) and the resulting mixture cooled to −70° C.Sulfuryl chloride (0.8 mL) was added and the mixture was stirred for 30min., after which imidazole (1.3 g) was added. The reaction mixture waswarmed to room temperature and stirred for an additional 2 hrs. Themixture was then diluted with methylene chloride and extracted withwater. The organic layer was concentrated to yield crude product MethylHydrogen 17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate,γ-Lactone (1.97 g). A small sample of the crude product was analyzed byHPLC. The analysis showed that the ratio of 9,11-olefin: 11,12-olefin:7,9-lactone was 75.5:7.2:17.3. When carried out at 0° C. but otherwiseas described above, the reaction yielded a product in which the9,11-olefin: 11,12-olefin: 7,9-lactone distribution was 77.6:6.7:15.7.This procedure combines into one step the introduction of a leavinggroup and elimination thereof for the introduction of the 9,11-olefinstructure of the enester, i.e., reaction with sulfuryl chloride causesthe 11α-hydroxy group of the hydroxy ester of Formula V to be replacedby halide and this is followed by dehydrohalogenation to the Δ^(9,11)structure. Thus formation of the enester is effected without the use ofa strong acid (such as formic) or a drying agent such as aceticanhydride. Also eliminated is the refluxing step of the alternativeprocess which generates carbon monoxide.

EXAMPLE 36A Scheme 1: Step 3C: Method D: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone

Hydroxyester (20 g) prepared as by Example 34, and methylene chloride(400 mL) were added to a clean dry three-neck round bottom flask fittedwith a mechanical stirrer, addition funnel and thermocouple. Theresulting mixture was stirred at ambient temperature until completesolution was obtained. The solution was cooled to 5° C. using an icebath. Methanesulfonyl chloride (5 mL) was added to the solution ofCH₂Cl₂ containing the hydroxyester, rapidly followed by the slowdropwise addition of triethylamine (10.8 mL). The addition rate wasadjusted so that the temperature of the reaction did not exceed 5° C.The reaction was very exothermic; therefore cooling was necessary. Thereaction mixture was stirred at about 5° C. for 1 h. When the reactionwas complete (HPLC and TLC analysis), the mixture was concentrated atabout 0° C. under 26 in Hg vacuum until it became a thick slurry. Theresulting slurry was diluted with CH₂Cl₂ (160 mL), and the mixture wasconcentrated at about 0° C. under 26 in Hg vacuum to obtain aconcentrate. The purity of the concentrate (mesylate product of FormulaIV wherein R³═H and —A—A— and —B—B— are both —CH₂—CH₂—, i.e., methylhydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone to methyl hydrogen17α-hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone was found to be 82% (HPLC area %). This material was used forthe next reaction without isolation.

Potassium formate (4.7 g), formic acid (16 mL) and acetic anhydride (8mL, 0.084 mol) were added to a clean dry reactor equipped withmechanical stirrer, condenser, thermocouple and heating mantle. Theresulting solution was heated to 70° C. and stirred for about 4-8 hours.The addition of acetic anhydride is exothermic and generated gas (CO),so that the rate of addition had to be adjusted to control bothtemperature and gas generation (pressure). The reaction time to preparethe active eliminating reagent was dependent on the amount of waterpresent in the reaction (formic acid and potassium formate containedabout 3-5% water each). The elimination reaction is sensitive to theamount of water present; if there is >0.1% water (KF), the level of the7,9-lactone impurity may be increased. This by product is difficult toremove from the final product. When the KF showed <0.1% water, theactive eliminating agent was transferred to the concentrate of mesylate(0.070 mol) prepared in the previous step. The resulting solution washeated to 95° C. and the volatile material was distilled off andcollected in a Dean Stark trap. When volatile material evolution ceased,the Dean Stark trap was replaced with the condenser and the reactionmixture was heated for additional 1 h at 95° C. Upon completion (TLC andHPLC analysis; <0.1% starting material) the content was cooled to 50° C.and vacuum distillation was started (26 in Hg/50° C.). The mixture wasconcentrated to a thick slurry and then cooled to ambient temperature.The resulting slurry was diluted with ethyl acetate (137 mL) and thesolution was stirred for 15 min. and diluted with water (137 mL). Thelayers were separated, and the aqueous lower layer was re-extracted withethyl acetate (70 mL). The combined ethyl acetate solution was washedonce with brine solution (120 mL) and twice with ice cold 1N NaOHsolution (120 mL each). The pH of aqueous was measured, and the organiclayer rewashed if the pH of the spent wash liquor was <8. When the pH ofthe spent wash was observed to be >8, the ethyl acetate layer was washedonce with brine solution (120 mL) and concentrated to dryness by rotaryevaporation using a 50° C. water bath. The resulting enester, solidproduct i.e., methyl hydrogen17α-hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-lactoneweighed 92 g (77% mol yield).

EXAMPLE 36B Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone

To a clean dry 250 mL three-neck round bottom flask fitted with amechanical stirrer, addition funnel and thermocouple was added 25 g(53.12 mmol) of the hydroxyester Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactoneprepared as by Example 34, followed by 150 mL of methylene chloride(Burdick & Johnson). The resulting mixture was stirred at ambienttemperature until a light slurry was obtained. The solution was cooledto −5° C. using an ice bath. Methanesulfonyl chloride (7.92 g, 69.06mmol) (Aldrich) was added to the solution of methylene chloridecontaining the hydroxyester, rapidly followed by the slow dropwiseaddition of triethylamine (7.53 g) (Aldrich). The addition rate wasadjusted so that the temperature of the reaction did not exceed 0° C.The reaction was very exothermic; therefore cooling was necessary.Addition time was 35 minutes. The reaction mixture was stirred at about0° C. for an additional 45 minutes. When the reaction was complete (lessthan 1% hydroxyester remaining indicated by HPLC and TLC analysis), themixture was concentrated by stripping approximately 110 mL to 125 mL ofthe methylene chloride solvent at atmospheric pressure. The reactortemperature reached approximately 40° C. to 45° C. during stripping.Where the reaction is not complete after the additional 45 minutes, anadditional 0.1 equivalent of methanesulfonyl chloride and an additional0.1 equivalent triethylamine can be charged to the reactor and thereaction checked for completion. The resulting mixture contained thecrude product methyl hydrogen17α-hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone. This material was used for the next reaction withoutisolation.

Anhydrous sodium acetate (8.7 g)(Mallinkrodt), glacial acetic acid (42.5mL) (Fisher) and acetic anhydride (0.5 mL) (Fisher) were added to asecond 250 mL clean dry reactor equipped with mechanical stirrer,condenser, thermocouple and heating mantle. The resulting solution washeated to 90° C. and stirred for about 30 minutes. The addition ofacetic anhydride is exothermic, so that the rate of addition had to beadjusted to control both temperature and pressure. Acetic anhydride wasadded to reduce the water content of the solution to an acceptable level(less than about 0.1%). When the KF showed <0.1% water, the acetic acidsolution was transferred to the concentrate of mesylate prepared asdiscussed in the first paragraph of this example. The temperature of theresulting mixture was about 55° C. to 60° C. after the transfer. Byusing acetic acid and sodium acetate instead of formic acid andpotassium formate as in Example 36A, gas generation was reduced.

The mixture was heated to 135° C. and maintained at that temperature forabout 60 to 90 minutes until volatile material evolution ceased. Thevolatile material distilled from the mixture was collected in a DeanStark trap. Upon completion (TLC and HPLC analysis; <0.1% startingmaterial) the heat source was removed. When the temperature of themixture reached 80° C., 150 mL of water was slowly added to the mixtureover 60 to 90 minutes. At the end of the water addition, the mixture hadcooled to a temperature of about 35° C. to 45° C. and a slurry had begunto form. The mixture was further cooled to 15° C. and maintained at thattemperature for about 30 to 60 minutes.

The mixture was filtered through a glass funnel. The filtrate was rinsedwith 100 mL of water. The filtrate was then washed a second time with anadditional 100 mL of water. The resulting filtrate was dried at 70° C.in vacuo to yield 25.0 g of dry product enester, methyl hydrogen17α-hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-lactone.HPLC assay indicated 70% of the desired 9,11-olefin, 15% of the11,12-olefin, and 5% of the 7,0-lactone.

This method had the beneficial result (relative to similar processes) of(i) reducing solvent volumes, (ii) reducing the number of separateoperational steps needed to produce the enester from the hydroxyester,(iii) reducing the washes needed, (iv) replacing extraction with waterprecipitation in isolating the final product, and (v) eliminating safetyconcerns previously associated with mixed anhydride formation and gasgeneration when formic acid is used instead of acetic acid.

EXAMPLE 37A Scheme 1: Step 3C: Method E: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone

Hydroxyester (100 g; 0.22 mol) prepared as by Example 34 was charged toa 2 L 3-neck round bottom flask equipped with mechanical stirrer,addition funnel, and thermocouple. A circulating cooling bath was usedwith automatic temperature control. The flask was dried prior toreaction because of the sensitivity of methanesulfonyl chloride towater.

Methylene chloride (1 L) was charged to the flask and the hydroxyesterdissolved therein under agitation. The solution was cooled to 0° C. andmethane sulfonyl chloride (25 mL; 0.32 mol) was charged to the flask viathe addition funnel. Triethylamine (50 mL; 0.59 mol) was charged to thereactor via the addition funnel and the funnel was rinsed withadditional methylene chloride (34 mL). Addition of triethylamine washighly exothermic. Addition time was around 10 min. under agitation andcooling. The charge mixture was cooled to 0° C. and held at thattemperature under agitation for an additional 45 min. during which thehead space of the reaction flask was flushed with nitrogen. A sample ofthe reaction mixture was then analyzed by thin layer chromatography andhigh performance liquid chromatography to check for reaction completion.The mixture was thereafter stirred at 0° C. for an additional 30 min.and checked again for reaction completion. Analysis showed the reactionto be substantially complete at this point; the solvent methylenechloride was stripped at 0° C. under 26″ mercury vacuum. Gaschromatography analysis of the distillate indicated the presence of bothmethane sulfonyl chloride and triethylamine. Methylene chloride (800 mL)was thereafter charged to the reactor and the resulting mixture wasstirred for 5 min. at a temperature in the range of 0-15° C. The solventwas again stripped at 0-5° C. under 26″ mercury vacuum yielding themesylate of Formula IV wherein R³ is H, —A—A— and —B—B— are —CH₂—CH₂—and R¹ is methoxy carbonyl. The purity of the product was about 90-95area %.

To prepare an elimination reagent, potassium formate (23.5 g; 0.28 mol),formic acid (80 mL) and acetic anhydride (40 mL) were mixed in aseparate dried reactor. Formic acid and acetic anhydride were pumpedinto the reactor and the temperature was maintained not greater than 40°C. during addition of acetic anhydride. The elimination reagent mixturewas heated to 70° C. to scavenge water from the reaction system. Thisreaction was continued until the water content was lower than 0.3% byweight as measured by Karl Fisher analysis. The elimination reagentsolution was then transferred to the reactor containing the concentratedcrude mesylate solution prepared as described above. The resultingmixture was heated to a maximum temperature of 95° C. and volatiledistillate collected until no further distillate was generated.Distillation ceased at about 90° C. After distillation was complete, thereaction mixture was stirred at 95° C. for an additional 2 hrs. andcompletion of the reaction was checked for thin layer chromatography.When the reaction was complete, the reactor was cooled to 50° C. and theformic acid and solvent removed from the reaction mixture under 26″mercury vacuum at 50° C. The concentrate was cooled to room temperatureand thereafter ethyl acetate (688 mL) was introduced and the mixture ofethyl acetate and concentrate stirred for 15 min. At this point, a 12%brine solution (688 mL) was introduced to assist in removing watersoluble impurities from the organic phase. The phases were then allowedto settle for 20 min. The aqueous layer was transferred to anothervessel to which an additional amount of ethyl acetate (350 mL) wascharged. This back extraction of the aqueous layer was carried out for30 min. after which the phases were allowed to settle and the ethylacetate layers combined. To the combined ethyl acetate layers, saturatedsodium chloride solution (600 mL) was charged and stirring carried outfor 30 min. The phases were then allowed to settle. The aqueous layerwas removed. An additional sodium chloride (600 mL) wash was carriedout. The organic phase was separated from the second spent wash liquor.The organic phase was then washed with 1 N sodium hydroxide (600 mL)under stirring for 30 min. The phases were settled for 30 min. to removethe aqueous layer. The pH of the aqueous layer was checked and it foundto be >7. A further wash was carried out with saturated sodium chloride(600 mL) for 15 min. The organic phase was finally concentrated under26″ mercury vacuum at 50° C. and the product recovered by filtration.The final product was a foamy brown solid when dried. Further drying at45° C. under reduced pressure for 24 hrs. yielded 95.4 g of the enesterproduct Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactonewhich assayed at 68.8%. The molar yield was 74.4% corrected for both thestarting hydroxy ester and the final enester.

EXAMPLE 37B Preparation of 7-methyl hydrogen17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7α,21-dicarboxylate

A reaction flask was charged with 5.5 g of the mesylate prepared in themanner of Example 23, 55 mL of 94.3% formic acid and 1.38 g of potassiumformate. The mixture was heated and stirred at reflux (104° C.) for twohours. At the end of the two hour period the formic acid was distilledunder reduced pressure. The residue was dissolved in ethyl acetate andwashed with 10% potassium carbonate (50 mL). The recovered aqueousportion was yellow in color. The ethyl acetate was washed with 5% sodiumhydroxide (50 mL). The aqueous portions were combined and acidified withdilute hydrochloric acid and the insoluble material extracted with ethylacetate. The ethyl acetate was evaporated to dryness under reducedpressure to give 1.0 g of a residue, 7-methyl hydrogen17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7α,21-dicarboxylate.

¹H NMR (CDCl₃) ppm 1.5 (s,3H), 1.4 (s,3H), 3.53 (s,3H), 5.72 (m,1H).

¹³C NMR (CDCl3) ppm 25.1 and 25.4 (18 CH₃ and 19 CH₃), 40.9 (10 C), 48.5(17 C), 51.4 (OCH₃), 118.4 (11 CH), 125.4 (4 CH), 132.4 (9 C), 138.5 and139.7 (13 C and 14 C), 168.2 (5 C), 172.4 (7 CO), 179.6 (22 CO), 198.9(3 CO).

EXAMPLE 37C Preparation of 7-methyl hydrogen5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate,γ-lactone

A reaction flask was charged with 5.5 g of the mesylate prepared in themanner of Example 23, 55 mL of 94.3% formic acid and 1.38 g of potassiumformate. The mixture was heated and stirred at reflux (104° C.) for twohours. At the end of the two hour period the formic acid was distilledunder reduced pressure. The residue was dissolved in ethyl acetate andwashed with 10% potassium carbonate (50 mL). The recovered aqueousportion was yellow in color. The ethyl acetate was washed with 5% sodiumhydroxide (50 mL). The ethyl acetate was evaporated to dryness underreduced pressure to give a 3.7 g residue. A 3.4 g portion of the residuewas chromatographed on 267 g of Merck silica gel (40-63μ). The productwas recovered with an elution scheme of ethyl acetate and toluene 37:63(v/v). After drying this product, 0.0698 g of a residue, 7-methylhydrogen 5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate,γ-lactone, was obtained.

¹H NMR (CDCl₃) ppm 1.03 (s,3H), 1.22 (s,3H), 3.70 (s,3H), 5.60(d,1H,J10), 5.98 (d,1H,J10).

MIR cm⁻¹ 2229 (CN), 1768 (lactone), 1710 (ester).

EXAMPLE 37D Isolation of9α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,bis(γ-lactone)

9α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,bis(γ-lactone) is a byproduct of the 11-mesylate elimination. A puresample was isolated from the reaction mixture of Example 37A viapreparative liquid chromatography followed by reverse phase preparativeHPLC. Thus, a 73 g residue was chromatographed over 2.41 kg of Mercksilica gel (40-63μ) with a gradient elution scheme of ethyl acetate andtoluene (20:80, 30:70, 40:60, 60:40, v/v). An enriched mixture (10.5 g)of the enamine and the 7,9-lactone was obtained in the 60:40 fractions.The progress of the purification was observed via TLC on EMF plates witha 60:40 (v/v) ethyl acetate, toluene eluent and visualization viasulfuric acid, SWUV. A portion (10.4 g) of the mixture was furtherpurified via reverse phase HPLC on Kromasil C8 (7μ) and a 30:70 (v/v)milliQ water and acetonitrile mobile phase. The 7,9-lactone (2.27 g) wasisolated as crystals from the mobile phase.

MIR cm⁻¹ 1762 (7,9-lactone and 17-lactone), 1677, 1622 (3-keto-Δ^(4,5)).

¹H NMR (CDCl₃) ppm 1.00 (s,3 H), 1.4 (s,3H), 2.05 (d,1H), 2.78 (d,1H),5.87 (s,1H).

¹³C NMR (CDCl₃) ppm 13.2, 19.0, 22.2, 23.2, 26.8, 28.8, 29.5, 30.8,33.1, 34.4, 35.1, 42.5, 43.6, 43.9, 45.0, 45.3, 89.9, 94.7, 129.1,161.5, 176.0, 176.4, 196.9.

Theory C, 71.85 and H, 7.34; Found C, 71.68 and H, 7.30.

EXAMPLE 37E Isolation of 7-methyl hydrogen5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate, γ-lactone

The compound 7-methyl hydrogen5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate, γ-lactonewas isolated after multiple preparative liquid chromatography on thereaction mixture obtained after elimination of the 11-mesyloxy group(Example 24). It was part of a cluster of less polar impurities asviewed via TLC on EMF plates with a 30:70 (v/v) ethyl acetate andmethylene chloride eluting system and visualized via sulfuric acid,SWUV. Generally, these less polar impurities were separated from thecrude enamine via preparative liquid chromatography. Specifically, 9.6 gof the crude enamine solution was chromatographed over 534 g of Mercksilica gel (40-63μ) using an ethyl acetate and toluene gradient elutionscheme (20:80, 30:70, 40:60, 60:40, v/v). The less polar impurities wereconcentrated in the 30:70 fractions. A 12.5 g pool of the less polarimpurities was collected in this fashion. This material was furtherchromatographed over 550 g of Merck silica gel (40-63μ) using an ethylacetate and methylene chloride gradient elution scheme (5:95, 10:90,20:80, 30:70, v/v). An enriched portion of the 20:80 fractions yielded1.2 g of residue. Additional chromatography of the 1.2 g residue over 53g of Merck silica gel (40-63μ) using an acetone and methylene chloridegradient (3:97, 6:94, 10:90, 15:85, v/v) yielded 0.27 g of 7-methylhydrogen 5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-dicarboxylate,γ-lactone from an enriched portion of the 10:90 fractions.

MS M+425, calculated for C₂₅H₃₁NO₅ (425.52).

MIR 2222 cm⁻¹ (nitrile), 1767 cm⁻¹ (lactone), 1727 cm⁻¹ (ester and3-ketone).

¹H NMR (CDCl₃) ppm 0.92 (s,3H), 1.47 (s,3H), 2.95 (m,1H), 3.65 (s,3H),5.90 (m,1H).

¹³C NMR (CDCl₃) ppm 14.0 (18 CH₃), 23.5 (15 CH₂), 27.0 (19 CH₃), 37.8,38.5 and 40.9 (7, 8 and 14 CH), 52.0 (OCH₃), 95.0 (17 C), 121.5 (23 CN),123.5 (11 CH), 135.3 (9 C), 174.2 and 176.2 (22 and 24 CO), 206 (3CO).

EXAMPLE 37F Preparation of 7-methyl hydrogen17-hydroxy-3-oxo-11α-(2,2,2-trifluoro-1-oxoethoxy)-17α-pregn-4-ene-7α,21-dicarboxylate

Hydroxyester (2.0 g, 4.8 mmols) prepared in the manner of Example 34 wasadded to 40 mL of methylene chloride in a clean, dry 3-neck, roundbottom flask equipped with a mechanical stirrer. Triethyl amine (0.61 g,6.10 mmols) and trifluoracetic anhydride (1.47 g, 7.0 mmols) were thenadded to the solution. This mixture was stirred at ambient temperatureovernight.

The mixture then was diluted with an additional 40 mL of methylenechloride. The mixture then was washed successively with 40 mL of water,40 mL of 1N HCl, and 40 mL of 1N NaOH solution. The resulting solutionwas then dried over magnesium sulfate and concentrated to dryness toafford 3.2 g of a light brown solid, 7-methyl hydrogen17-hydroxy-3-oxo-11α-(2,2,2-trifluoro-1-oxoethoxy)-17α-pregn-4-ene-7α,21-dicarboxylate.

The residue was further analyzed and purified by chromatography. HPLCconditions: column—Waters Symmetry C18 (150 mm×4.6 mm i.d., 5 micronparticle size); column temperature—ambient; mobilephase—acetonitrile/water, 30/70 by volume; flow rate—1.0 mL/minute;injection volume—20 microliters; sample concentration—1.0 mg/ml;detection—UV at 210 nm; pressure—1500 psi; and run time—45 minutes. TLCconditions: adsorbent—Merck Silica Gel 60 F₂₅₄; solvent system—ethylacetate/toluene, 65/35 by volume; visualization technique—shortwave; andapplication amount—100 micrograms.

EXAMPLE 37G Preparation of 7-methyl hydrogen11α-(acetyloxy)-17-hydroxy-3-oxo-17α-pregn-4-ene-7,21-dicarboxylate,γ-lactone

Hydroxyester (2.86 g, 6.87 mmole) prepared in the manner of Example 34was added to 15 mL of methylene chloride in a clean, dry 3-neck, roundbottom flask equipped with a mechanical stirrer. Triethyl amine (1.39 g,13.7 mmol), dimethylaminopyridine (0.08 g, 0.6 mmol) and aceticanhydride (1.05 g, 10.3 mmol) were then added to the solution. Thismixture was stirred at ambient temperature overnight.

The mixture then was diluted with 150 mL of ethyl acetate and 25 mL ofwater. This ethyl acetate solution then was washed 25 mL of citric acidsolution. The solution was then dried over magnesium sulfate andconcentrated to dryness to afford 3.33 g of a light brown solid,7-methyl hydrogen11α-(acetyloxy)-17-hydroxy-3-oxo-17α-pregn-4-ene-7,21-dicarboxylate,γ-lactone.

The residue was further analyzed and purified by chromatography. HPLCconditions: column—Waters Symmetry C18 (150 mm×4.6 mm i.d., 5 micronparticle size); column temperature—ambient; mobilephase—acetonitrile/water, 30/70 by volume; flow rate—1.0 mL/minute;injection volume—20 microliters; sample concentration—1.0 mg/mL;detection—UV at 210 nm; pressure—1500 psi; and run time—45 minutes. TLCconditions: adsorbent—Merck Silica Gel 60 F₂₅₄; solvent system—methylenechloride/methanol, 95/5 by volume; visualization technique—shortwave;and application amount—100 micrograms.

EXAMPLE 37H Scheme 1: Step 3C: Method F: Preparation of 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate,γ-lactone

Potassium formate (1.5 g, 0.018 mol), formic acid 60 mL, 1.6 mol) andacetic anhydride (29.5 mL, 0.31 mol) were added to a clean, dry 250 mLreactor equipped with a mechanical stirrer, condenser, thermocouple andheating mantle. The solution was then was stirred at 70° C. for 4 hoursand cooled to ambient temperature to provide an elimination reagentuseful for converting the mesylate of Formula IV to the product of thisexample.

The preformed TFA/TFA anhydride elimination reagent was added to 70.0 g(0.142 mol) of the mesylate prepared in the manner of Example 23. Theresulting mixture was heated to 95° C. to 105° C. for 2.5 hrs., thedegree of conversion being periodically checked by TLC or HPLC. Theresulting residue was cooled to 50° C., diluted with ice water (1.4 L)and stirred for 1 hour. The mixture was left standing overnight atambient temperature. The layers were separated and aqueous phase wasre-extrated with ethyl acetate (75 mL). The ethyl acetate solution wasthen successively washed with a water/brine mixture (70 mL), anotherwater/brine mixture (60 mL), 1N sodium hydroxide (60 mL), and a thirdwater/brine mixture (60 mL). Brine strength was 12% by weight. The ethylacetate solution was then dried over sodium sulfate, filtered andconcentrated to dryness by rotary evaporator to afford a 4.5 g mixtureof both the desired product and an unknown impurity. The ratio of theimpurity/product by HPLC area was about 50/15 respectively. The majorproduct from this reaction was the impurity which was identified as7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone.

The mixture was purified by column chromatography to afford 1.9 g ofanalytically pure 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone.

The residue was further analyzed and purified by chromatography. HPLCconditions: column—Waters Symmetry C18 (150 mm×4.6 mm i.d., 5 micronparticle size); column temperature—ambient; mobilephase—acetonitrile/water, 30/70 by volume; flow rate—1.0 mL/minute;injection volume—20 microliters; sample concentration—1.0 mg/mL;detection—UV at 210 nm; pressure—1500 psi; and run time—45 minutes. TLCconditions: adsorbent—Merck Silica Gel 60 F₂₅₄; solventsystem—chloroform/methyl t-butyl ether/isopropanol, 70/28/2 by volume;visualization technique—50% by volume aqueous H₂SO₄/LWUV and 50% byvolume H₂SO₄/phosphomolybdic acid; and application amount—100micrograms.

EXAMPLE 37I Preparation of 7-methyl hydrogen17-hydroxy-3,11-dioxo-17α-pregn-4-ene-7α,21-dicarboxylate, γ-lactone

A Jones Reagent was prepared by dissolving 6.7 g of chromic anhydride(CrO₃) in 6 mL of concentrated sulfuric acid and carefully diluting thatmixture with distilled water to 50 mL. One mL of this reagent issufficient to oxidize 2 mmol of a secondary alcohol to a ketone.

Hydroxyester (10.0 g, 24.0 mmole) prepared in the manner of Example 34was dissolved/suspended in 1200 mL of acetone. To this mixture was added8.992 mL of the Jones Reagent and the combined mixture was stirred for10 minutes. An aliquot of the reaction mixture, after being treated withwater and extracted with methylene chloride, was analyzed by HPLC(column: Beckman Ultrasphere ODS C18, 4.6 mm×250 mm, 5 micron; solventgradient: acetonitrile/water=1/99 to 100/0 in 20 minutes at a flow rateof 1.5 mL/minute; detector: UV210 nm). The reaction was complete asevidenced by the lack of any significant amount of the starting materialin the reaction mixture. The HPLC retention time for the startingmaterial (hydroxester) is 13.37 minute and for the product ketone was14.56 min.

The reaction was worked up by adding 200 mL of water and 300 mL ofmethylene chloride. The organic layer was separated from the aqueouslayer and washed again with 200 mL of water. The organic layer wasseparated from the aqueous layer and dried over magnesium sulfate. Thesolvent was evaporated to provide 9.52 g of the off-white solid (95.6%crude yield) 7-methyl hydrogen17-hydroxy-3,11-dioxo-17α-pren-4-ene-7α,21-dicarboxylate, γ-lactone.

The structure assignment was based on the mass spectrum (m/e 414), HNMR(DMF-d7) and CNMR (DMF-d7). In HNMR, the characteristic peak of the 11-H(4.51 ppm, doublet, j=5.8 Hz) found in the hydroxester was absent. InCNMR, a peak appeared at 208.97 ppm which is expected for the 11-ketocarbon.

CNMR (400 MHz, DMF-d7) 208.97 (11-keto), 197.70 (3-keto), 176.00(22-lactone), 173.34 (7-COOMe), 167.21 (C5), 125.33 (C4), 93.63 (C17),and other peaks in the region of 15 to 57 ppm.

EXAMPLE 37J Preparation of dimethyl11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate, γ-lactone

A solution of 3.5 g (8.4 mmols) of the hydroxyester prepared in themanner of Example 34 in 42 mL of methanol was mixed with 4 mL ofmethanolic 4N potassium hydroxide (8 mmols). The slurry was stirred atroom temperature overnight and heated to reflux for one hour. Themethanol was evaporated under vacuum and the residue mixed with 50 mL ofethyl acetate. The ethyl acetate was evaporated under vacuum and theresidue digested with 50 mL of ethyl acetate. The dried solid wascombined with 50 mL of acetone and 2 mL of methyl iodide (32.1 mmols).The mixture was stirred at room temperature for 18 hours. During thistime most of the solids dissolved. The mixture was filtered and thefiltrate evaporated to dryness under vacuum. The residue was digestedwith ethyl acetate, the solids then removed via filtration and thesolvent removed under vacuum distillation. The residue was determined tobe a 78:22 (v/v) mixture of dimethyl11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate, γ-lactoneand the starting material hydroxyester via H¹ NMR. This mixture wasadequate for use as an HPLC marker without further purification.

¹H NMR (CDCl₃) indicated the following features: ppm 0.93 (s,3H), 1.37(s,3H), 3.64 (s,3H) 3.69 (s,3H).

EXAMPLE 37K Preparation of11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate, γ-lactone

To 11.86 g (28.5 mmol) of the hydroxyester prepared in the manner ofExample 34 was added 50 mL of methanol and 20 mL of 2.5 M NaOH. Thesuspension was heated to reflux. After 25 minutes, a portion of thestarting ester remained unreacted as judged by HPLC (Zorbax SB-C8150×4.6 mm, 2 ml/min., linear gradient 35:65 A:B to 45:55 A:B over 15min, A=acetonitrile/methanol 1:1, B=water/0.1% trifluoroacetic acid,detection at 210 nm) and 10 mL of 10 M NaOH was added. After 1.5 hours,a trace of ester remained unreacted as judged by HPLC. The reactionmixture was allowed to stand at about 25 degrees for 64 hours.

The mixture was diluted with 100 mL of water and then made stronglyacidic with 20 mL of concentrated HCl. The resulting gummy precipitatewas stirred until the precipitate became a suspension. The solid wasisolated by filtration, resuspended in methanol and filtered to give3.75 g of a brown solid. The material was dissolved in 8 mL of hot DMFand the mixture was diluted with 40 mL of methanol. The acidcrystallized and was isolated by filtration to give 1.7 g of a fluffywhite solid, 11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone.

1H-nmr (400 MHz, deuterodimethyl sulfoxide) d 0.80 (s, 3H), 1.25 (s,3H), 1.2-2.7 (m, 20H), 3.8 (brs, 1H), 4.45 (m, 1H), 5.50 (s, 1H). Thecarboxyl proton was not observed due to the presence of an HOD peak at3.4 ppm.

EXAMPLE 38 Scheme 1: Step 3C: Method G: Preparation of 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone

The procedure of Example 37A was repeated except that the multiplewashing steps were avoided by treating the reaction solution with an ionexchange resin, basic alumina or basic silica. Conditions for treatmentwith basic alumina or basic silica are set forth in Table 38. Each ofthese treatments was found effective for removal of impurities withoutthe multiple washes of EXAMPLE 44 to produce 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone.

TABLE 38 Factor Set point Purpose of Experiment Key results Basic 2g/125 g Treating the reaction mixture The yield alumina product withbasic alumina to remove was 93% Et₃N.HCl salt and to eliminate the 1 NNaOH and 1 N HCl washes Basic 2 g/125 g Treating the reaction mixtureThe yield silica product with basic silica which is was 95% cheaper toremove Et₃N.HCl salt and eliminate 1 N NaOH and 1 N HCl washes

EXAMPLE 39 Scheme 1: Step 3C: Method H: Preparation of 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone

Potassium acetate (4 g) and trifluoroacetic acid (42.5 mL) were mixed ina 100 mL reactor. trifluoroacetic anhydride (9.5 mL) was added to themixture at a rate controlled to maintain temperature during additionbelow 30° C. The solution was then heated to 30° C. for 30 min. toprovide an elimination reagent useful for converting the mesylate ofFormula IV to the enester of Formula II.

The preformed TFA/TFA anhydride elimination reagent was added to asolution of the mesylate of Formula IV previously prepared as in Example37A. The resulting mixture was heated at 40° C. for 4½ hrs., the degreeof conversion being periodically checked by TLC or HPLC. When thereaction was complete, the mixture was transferred to a 1-neck flask andconcentrated to dryness under reduced pressure at room temperature (22°C.). Ethyl acetate (137 mL) was added to the mixture to obtain completedissolution of solid phase material after which a water/brine mixture(137 mL) was added and the resulting two phase mixture stirred for 10min. The phases were then allowed to separate for 20 min. Brine strengthwas 24% by weight. The aqueous phase was contacted with an additionalamount of ethyl acetate (68 mL) and the two phase mixture thus preparedwas stirred for 10 min. after which it was allowed to stand for 15 min.for phase separation. The ethyl acetate layers from the two extractionswere combined and washed with 24% by weight brine (120 mL), anotheraliquot of 24% by weight brine (60 mL), 1 N sodium hydroxide solution(150 mL) and another portion of brine (60 mL). After each aqueous phaseaddition, the mixture was stirred for 10 min. and allowed to stand for15 min. for separation. The resulting solution was concentrated todryness under reduced pressure at 45° C. using a water aspirator. Thesolid product (8.09 g) was analyzed by HPLC and found to include 83.4area % of the enester 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone;2.45 area % of the 11,12-olefin; 1.5% of the 7,9-lactone; and 1.1% ofunreacted mesylate.

EXAMPLE 40 Scheme 1: Step 3C: Method I: Preparation of 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone

The mesylate having the structure prepared per Example 23 (1.0 g),isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) were placedin a 50 ml flask and heated to 90° C. with stirring. After 5 hours themixture was cooled to 25° C. and concentrated in vacuo at 10 mm of Hg.The residue was dissolved in CH₂Cl₂ (20 ml) and washed with 5% aqueousNaHCO₃. The CH₂Cl₂ layer was concentrated in vacuo to give 1.47 g of atan oil. This material was recrystallized from CH₂Cl₂/Et₂O to give 0.50g of enol acetate of Formula IV(Z).

This material was added to a mixture of sodium acetate (0.12 g) andacetic acid (2.0 ml) that had been previously heated to 100° C. withstirring. After 60 minutes the mixture was cooled to 25° C. and dilutedwith CH₂Cl₂ (20 ml). The solution was washed with water (20 ml) anddried over MgSO₄. The drying agent was removed by filtration and thefiltrate was concentrated in vacuo to give 0.4 g of the desired9,11-olefin, 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone.The crude product contained less than 2% of the 7,9-lactone impurity.

EXAMPLE 41 Thermal Elimination of Mesylate in DMSO Scheme 1: Step 3C:Method J: Preparation of 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone

A mixture of 2 g of mesylate and 5 ml of DMSO in a flask was heated at80° C. for 22.4 hours. HPLC analysis of the reaction mixture indicatedno starting material was detected. To the reaction was added water (10ml) and the precipitate was extracted with methylene chloride threetimes. The combined methylene chloride layers were washed with water,dried over magnesium sulfate, and concentrated to give the enester7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-dicarboxylate, γ-lactone.

EXAMPLE 42 Scheme 1: Step 3D: Method B: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

In a 50 mL pear-shaped flask under stirring the enester of Formula IIA(1.07 g assaying 74.4% enester), trichloroacetamide (0.32 g),dipotassium hydrogen phosphate (0.70 g) as solid were mixed withmethylene chloride (15.0 mL). Hydrogen peroxide (30% by weight; 5.0 mL)was added via a pipet over a 1 min. period. The resulting mixture wasstirred for 6 hrs. at room temperature at which point HPLC analysisshowed that the ratio of epoxymexrenone to enester in the reactionmixture was approximately 1:1. Additional trichloroacetamide (0.32 g)was added to the reaction mixture and reaction continued under agitationfor 8 more hours after which time the remaining proportion of enesterwas shown to have been reduced to 10%. Additional trichloroacetamide(0.08 g) was added and the reaction mixture was allowed to standovernight at which point only 5% of unreacted enester remained relativeto epoxymexrenone in the mixture.

EXAMPLE 43 Scheme 1: Step 3D: Method C: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Enester of Formula IIA (5.4 g, assaying 74.4% enester) was added to a100 mL reactor. Trichloroacetamide (4.9 g) and dipotassium hydrogenphosphate (3.5 g) both in solid form were added to the enester followedby methylene chloride (50 mL). The mixture was cooled to 15° C. and a30% hydrogen peroxide (25 g) was added over a ten min. period. Thereaction mixture was allowed to come to 20° C. and stirred at thattemperature for 6 hrs., at which point conversion was checked by HPLC.Remaining enester was determined to be less than 1% by weight.

The reaction mixture was added to water (100 mL), the phases wereallowed to separate, and the methylene chloride layer was removed.Sodium hydroxide (0.5 N; 50 mL) was added to the methylene chloridelayer. After 20 min. the phases were allowed to separate HCl (0.5 N; 50mL) was added to the methylene chloride layer after which the phaseswere allowed to separate and the organic phase was washed with saturatedbrine (50 mL). The methylene chloride layer was dried over anhydrousmagnesium sulfate and the solvent removed. A white solid (5.7 g) wasobtained. The aqueous sodium hydroxide layer was acidified and extractedand the extract worked up to yield an additional 0.2 g of product. Yieldof epoxymexrenone was 90.2%.

EXAMPLE 44 Scheme 1: Step 3D: Method D: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Enester of Formula IIA was converted to epoxymexrenone in the mannerdescribed in Example 43 with the following differences: the initialcharge comprised of enester (5.4 g assaying 74.4% enester),trichloroacetamide (3.3 g), and dipotassium hydrogen phosphate (3.5 g).Hydrogen peroxide solution (12.5 mL) was added. The reaction wasconducted overnight at 20° C. after which HPLC showed a 90% conversionof enester to epoxymexrenone. Additional trichloroacetamide (3.3 g) and30% hydrogen peroxide (5.0 mL) was added and the reaction carried outfor an additional 6 hrs. at which point the residual enester was only 2%based on the enester charge. After work up as described in Example 43,5.71 g of epoxymexrenone resulted.

EXAMPLE 45 Scheme 1: Step 3D: Method E: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

The enester of Formula IIA was converted to epoxymexrenone in the mannergenerally described in Example 43. In the reaction of this Example,enester charge was 5.4 g (assaying 74.4% enester), thetrichloroacetamide charge was 4.9 g, hydrogen peroxide charge was 25 g,dipotassium hydrogen phosphate charge was 3.5 g. The reaction was run at20° C. for 18 hrs. The residual enester was less than 2%. After work up,5.71 g of epoxymexrenone resulted.

EXAMPLE 46 Scheme 1: Step 3D: Method F: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Enester of Formula IIA was converted to epoxymexrenone in the mannerdescribed in Example 43 except that the reaction temperature in thisExample was 28° C. The materials charged in the reactor included enester(2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7g), hydrogen peroxide (17.0 g) and methylene chloride (50 mL). After 4hrs. reaction, unreacted enester was only 2% based on the enestercharge. After work up as described in Example 43, 3.0 g ofepoxymexrenone was obtained.

EXAMPLE 47-1 Scheme 1: Step 3D: Method G: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Enester of Formula IIA (40.0 g, assaying 68.4% enester) was charged intoa 1000 mL, jacketed reactor and dissolved in 175 mL of methylenechloride. The solution was stirred as trichloroacetamide (22.3 g) anddipotassium hydrogen phosphate (6.0 g) were added as solids. The mixturewas stirred at 400 RPM and the temperature adjusted to 27° C. with aconstant temperature bath to control the liquid circulating through thereactor jacket. Hydrogen peroxide (72.8 mL at 30% assay) was added overa 3 to 5 minute period. Following the hydrogen peroxide addition, themixture was stirred at 400 RPM and 27° C. HPLC assay indicated that thereaction was 99% complete within 5 hours. At the end of six hours, 72.8mL of water was added. The aqueous hydrogen peroxide was separated andback extracted one time with 50 mL of methylene chloride. The combinedmethylene chloride was washed with 6% sodium sulfite (62.3 mL) todestroy any contained peroxide. The methylene chloride removal wasstarted with atmospheric distillation and concluded under vacuum. Ayellowish residue (48.7 g, 55.4% assay) was obtained. This correlatedwith a 94.8% assay adjusted molar yield.

A portion (47.8 g) of the residue was combined with 498 mL of ethanol 3A(95% ethanol denatured with 5% methanol). The mixture was heated toreflux and 249 mL of distillate removed at atmospheric pressure. Themixture was cooled to 25° C. and filtered. An ethanol 3A rinse (53 mL)was used to assist the transfer. The dried solid weighed 27.6 g (87.0%assay) which correlated with a 91% recovery. A portion of the solid(27.0 g) was dissolved in 292 mL of methyl ethyl ketone at reflux. Thehot solution was filtered through a pad of solka floc (powderedcellulose) with another 48.6 mL of methyl ethyl ketone used to assistthe transfer. A 146 mL portion of the methyl ethyl ketone was removedvia atmospheric distillation. The solution was cooled to 50° C. andstirred for one hour as the product crystallized. After one hour themixture was cooled to 25° C. Stirring was continued for one hour and thesolid filtered with 48.6 mL of methyl ethyl ketone used as a rinse. Thesolid was dried to a constant weight of 20.5 g which represented an87.2% recrystallization recovery. The reaction yield and ethanol, methylethyl ketone recoveries combined for a 75% overall yield.

The methyl ethyl ketone mother liquor was suitable for recycling with anincoming methylene chloride solution from a subsequent reaction. Thecombined methylene chloride and methyl ethyl ketone mixture wasevaporated to dryness with atmospheric and vacuum distillation. Theresidue was combined with 19 volumes of ethanol 3A based onepoxymexrenone content. One half of the solvent was removed underatmospheric distillation. After cooling to 25° C. the solid was filteredand dried. The dry solid was dissolved in 12 volumes of methyl ethylketone at reflux. The hot solution was filtered through a solka floc padwith 2 volumes of methyl ethyl ketone added as a rinse. The filtrate wasconcentrated with the atmospheric distillation of 6 volumes of methylethyl ketone. The solution was cooled to 50° C. and stirred for one houras the product crystallized. After one hour the mixture was cooled to25° C. Stirring was continued for one hour and the solid filtered with 2volumes of methyl ethyl ketone used as a rinse. The solid was dried to aconstant weight. The incorporation of the methyl ethyl ketone motherliquor raised the overall yield to 80-85%.

This method appears particularly suited for scaleup since it maximizesthroughput and minimizes washing volumes and waste.

EXAMPLE 47A Scheme 1: Step 3D: Method H: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

Enester of Formula IIA (17 g, assaying 72% enester) was dissolved inmethylene chloride (150 mL) after which trichloroacetamide (14.9 g) wasadded under slow agitation. The temperature of the mixture was adjustedto 25° C. and a solution of dipotassium hydrogen phosphate (10.6 g) inwater (10.6 mL) was stirred into the enester substrate solution under400 rpm agitation. Hydrogen peroxide (30% by weight solution; 69.4 mL)was added to the substrate/phosphate/trichloroacetamide mixture over a3-5 min. period. No exotherm or oxygen evolution was observed. Thereaction mixture thus prepared was stirred at 400 rpm and 25° C. for18.5 hrs. No oxygen evolution was observed throughout the course of thereaction, but analysis of the hydrogen peroxide consumption indicatedthat some oxygen was formed during the reaction. The reaction mixturewas diluted with water (69.4 mL) and the mixture stirred at about 250rpm for 15 min. No temperature control was necessary for this operationand it was conducted essentially at room temperature (any temperature inthe range of 5-25° C. being acceptable). The aqueous and organic layerswere allowed to separate and the lower methylene chloride layer wasremoved.

The aqueous layer was back extracted with methylene chloride (69.4 mL)for 15 min. under agitation of 250 rpm. The layers were allowed toseparate and the lower methylene chloride layer was removed. The aqueouslayer (177 g; pH=7) was submitted for hydrogen peroxide determination.The result (12.2%) indicated that 0.0434 mol of hydrogen peroxide wereconsumed in the reaction of 0.0307 mol of olefin. The excess hydrogenperoxide consumption was a measure of oxygen generation in the reaction.Back extraction with a small amount of methylene chloride volume wassufficient to insure no loss of epoxymexrenone in the aqueous layer.This result was confirmed with the application of a second largemethylene chloride extraction in which only trichloroacetamide wasrecovered.

The combined methylene chloride solutions from the above describedextractions were combined and washed with 3% by weight sodium sulfitesolution (122 mL) for at least 15 min. at about 250 rpm. A negativestarch iodide test (KI paper; no color observed; in a positive test apurple coloration indicates the presence of peroxide) was observed atthe end of the stir period.

The aqueous and organic layers were allowed to separate and the lowermethylene chloride layer removed. The aqueous layer (pH=6) wasdiscarded. Note that addition of sodium sulfite solution can cause aslight exotherm so that such addition should be carried out undertemperature control.

The methylene chloride phase was washed with 0.5 N sodium hydroxide (61mL) for 45 min. at about 250 rpm and a temperature in the range of15-25° C. (pH=12-13). Impurities derived from trichloroacetamide wereremoved in this process. Acidification of the alkaline aqueous fractionfollowed by extraction of the methylene chloride confirmed that verylittle epoxymexrenone was lost in this operation.

The methylene chloride phase was washed once with 0.1 N hydrochloricacid (61 mL) for 15 min. under 250 rpm agitation at a temperature in therange 15-25° C. The layers were then allowed to separate and the lowermethylene chloride layer removed and washed again with 10% by weightaqueous sodium chloride (61 mL) for 15 min at 250 rpm at a temperaturein the range of 15-25° C. Again the layers were allowed to separate andthe organic layer removed. The organic layer was filtered through a padof Solkafloc and then evaporated to dryness under reduced pressure.Drying was completed with a water bath temperature of 65° C. Anoff-white solid (17.95 g) was obtained and submitted for HPLC assay.Epoxymexrenone assay was 66.05%. An adjusted molar yield for thereaction was 93.1%.

The product was dissolved in hot methyl ethyl ketone (189 mL) and theresulting solution was distilled at atmospheric pressure until 95 mL ofthe ketone solvent had been removed. The temperature was lowered to 50°C. as the product crystallized. Stirring was continued at 50° C. for 1hr. The temperature was then lowered to 20-25° C. and stirring continuedfor another 2 hrs. The solid was filtered and rinsed with MEK (24 mL)and the solid dried to a constant weight of 9.98 g, which by HPLC assaycontain 93.63% epoxymexrenone. This product was re-dissolved in hot MEK(106 mL) and the hot solution filtered through a 10 micron line filterunder pressure. Another 18 mL of MEK was applied as a rinse and thefiltered MEK solution distilled at atmospheric pressure until 53 mL ofsolvent had been removed. The temperature was lowered to 50° C. as theproduct crystallized; and stirring was continued at 50° C. for 1 hr. Thetemperature was then lowered to 20-25° C. and held at that temperaturewhile stirring was continued for another 2 hrs. The solid product wasfiltered and rinsed with MEK (18 mL). The solid product was dried to aconstant weight of 8.32 g which contained 99.6% epoxymexrenone perquantitative HPLC assay. The final loss on drying was less than 1.0%.Overall yield of epoxymexrenone in accordance with the reaction and workup of this Example is 65.8%. This overall yield reflected a reactionyield of 93%, an initial crystallization recovery of 78.9%, and arecrystallization recovery of 89.5%.

EXAMPLE 47B Preparation of 7-methyl hydrogen11α,12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone

The Δ^(11,12) olefin of the enester is a byproduct of the 11-mesylateelimination. A pure sample was isolated from a a reaction mixtureprepared in the the manner of Example 37A via repetitive preparativeliquid chromatography. Thus, a 73 g residue (prepared as described inExample 37A) was chromatographed over 2.41 kg of Merck silica gel(40-63μ) with an ethyl acetate, toluene gradient elution scheme (20:80,30:70, 40:60, 60:40, v/v). Enriched Δ^(11,12) olefin portions werecombined from selected 30:70 fractions. TLC on EMF plates using ethylacetate/toluene 60:40 (v/v) with sulfuric acid SWUV visualization servedas a guide for choosing the appropriate fractions. The 7.9 g of crudeΔ^(11,12) olefin (80 area % via HPLC) obtained after the removal ofsolvent was chromatographed over 531 g of Merck silica gel (40-63μ) withan ethyl acetate/methylene chloride gradient elution scheme (10:90,20:80, 35:65, v/v). Pure 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,11-diene-7α,21-dicarboxylate, γ-lactone(3.72 g) was obtained from selected 20:80 fractions. The selection offractions was based on TLC evaluation as in the previous situation.

MIR cm⁻¹ 1767 (lactone), 1727 (ester), 1668 and 1616 (3-Keto-Δ^(4,5)).

¹H NMR (CDCl₃) ppm 1.05 (s,3H), 1.15 (s,3H), 3.66 (s,3H), 5.58 (dd,1H),5.80 (s,1H), 5.88 (dd,1H).

¹³C NMR (CDCl₃) ppm 17.41, 18.58, 21.73, 28.61, 32.28, 33.63, 34.91,35.64, 35.90, 38.79, 42.07, 44.12, 48.99, 49.18, 51.52, 93.81, 126.43,126.69, 133.76, 166.24, 172.91, 176.64, 198.56.

A solution of 1.6 g (3.9 mmols) of 7-methyl hydrogen17-hydroxy-3-oxo-17α-pregna-4,11-diene-7α,21-dicarboxylate, γ-lactone in16 mL of methylene chloride was mixed with 2.2 mL oftrichloroacetonitrile (22.4 mmols) and 0.75 g of dipotassium phosphate(4.3 mmols). The mixture was stirred and combined with 6.7 mL of 30%hydrogen peroxide (66 mmols). Stirring was continued at 25° C. for 45hours. At the end of this time 28 mL of methylene chloride and 39 mL ofwater were added. The organic portion was isolated and washed insuccession with a) 74 mL of 3% sodium sulfite, b) 62 mL of 1 N sodiumhydroxide, c) 74 mL of 1 N hydrochloric acid, and d) 31 mL of 10% brine.The organic portion was separated again, dried over magnesium sulfate,and evaporated to dryness under vacuum. The 1.25 g residue waschromatographed over 138.2 g of Merck silica gel (40-63μ) using amethyl-t-butyl ether and toluene gradient system (40:60, 60:40, 75:25,v/v). Appropriate portions of the 60:40 and 75:25 fractions werecombined after TLC evaluation to give 0.66 g of pure 7-methyl hydrogen11α,12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone. The TLC system utilized EMF plates and a 75:25 (v/v)methyl-t-butyl ether and toluene elution scheme with sulfuric acid andSWUV for visualization.

¹H NMR (CDCl₃) ppm 1.09 (s,3H), 1.30 (s,3H) 3.05 (AB^(11,12) 2H for),3.67 (s,3H), 5.80 (s,1H).

¹³C NMR (CDCl₃) ppm 14.2, 18.0, 21.2, 28.8, 31.9, 33.5, 34.6, 34.7,35.1, 35.5, 37.4, 38.3, 41.8, 46.0, 47.2, 50.4, 51.7, 56.7, 94.0, 126.7,165.2, 172.5, 176.7, 198.1.

Theory: C, 69.54 and H, 7.30; Found: C, 69.29 and H, 7.17.

EXAMPLE 47C Isolation of 7-methyl hydrogen4α,5α:9α,11α-diepoxy-17-hydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone

Crude epoxymexrenone (157 g) prepared from 200 g of the enester in themanner of Example 26 was subjected to chromatography over 4.4 kg ofMerck silica gel (40-63μ). An 88.1 g portion was recovered with an isacetonitrile and toluene 10:90 (v/v) elution scheme. The isolated solidwas dissolved in 880 mL of hot methyl ethyl ketone and filtered througha pad of solka floc. Another 88 mL of methyl ethyl ketone was applied asa rinse. The filtrate was concentrated via the removal of 643 mL ofsolvent and the mixture cooled to room temperature. The solids werefiltered and rinsed with methyl ethyl ketone. After drying, 60.2 g ofepoxymexrenone assaying 96.8% via HPLC was obtained. The filtrate wasconcentrated to dryness under reduced pressure. The 9.3 g residue wasrecrystallized from 99 mL of methyl ethyl ketone to yield 2.4 g of drysolid. A 400 mg portion of the solid was subjected to reverse phasepreparative HPLC on a YMC ODS AQ column. Pure 7-methyl hydrogen4α,5α:9α,11α-diepoxy-17-hydroxy-3-oxo-17α-pregnane-7α,21-dicarboxylate,γ-lactone (103 mg) was isolated with an elution scheme of acetonitrile(24%), methanol (4%) and water (72%).

¹H NMR (CDCl₃) ppm 0.98 (s,3H), 1.32 (s,3H), 2.89 (m,1 H), 3.07(s,d,2H), 3.73 (s,3H).

MS, M+430, calculated for C₂₄H₃₀O₇(430.50).

EXAMPLE 47D Isolation of 7-methyl hydrogen17-hydroxy-3,12-dioxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone

A methyl ethyl ketone mother liquor obtained in the manner of Example 26was evaporated to dryness under reduced pressure. A 4.4 g portion of theresidue was subjected to chromatography on 58.4 g of BTR Zorbax LP(40μ). Elution with a gradient of methyl ethyl ketone and methylenechloride (25:75 to 50:50, v/v) yielded 1.38 g of material. A 1.3 gportion of this material was further purified via reverse phasepreparative HPLC using acetonitrile (30%), methanol (5%) and water (65%)as the mobile phase and a YMC ODS AQ column (10μ). The product wasobtained from the enriched fractions via methylene chloride extraction.The methylene chloride was evaporated to dryness and the 175 mg residuerepurified via reverse phase preparative HPLC using acetonitrile (24%),methanol (4%) and water (72%) as the mobile phase and a YMC ODS AQcolumn. Methylene chloride extraction of enriched fractions yielded 30.6mg of pure 7-methyl hydrogen17-hydroxy-3,12-dioxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone.

¹H NMR (CDCl₃) ppm 1.17 (s,3H), 1.49 (s,3H), 3.13 (m,1H), 3.62 (s,3H),5.77 (s,1H), 5.96 (s,1H).

¹³C NMR (CDCl₃) ppm 13.1, 21.0, 28.0, 29.4, 33.1, 33.4, 33.9, 35.5,36.7, 40.3, 41.5, 43.0, 43.4, 52.0, 55.0, 91.0, 123.7, 126.7, 163.2,167.9, 171.8, 176.8, 197.4, 201.0.

EXAMPLE 47E Preparation of9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,dihydrate, dipotassium salt

A suspension containing 2.0 g (4.8 mmol) of epoxymexrenone prepared inthe manner of Example 43, 10 mL of water, 3 mL of dioxane and 9.3 mL of1.04 N aqueous potassium hydroxide (9.7 mmol) was prepared. The mixturewas stirred at 25° C. for 3 hours. A yellow, homogenous solution wasformed during the first two hours. The temperature was raised to 70° C.and stirring continued for an additional 3 hours. The solvent wasremoved via vacuum distillation and the residue purified via reversephase chromatography over 90 g of C18 silica gel using water as theeluent. The desired fractions were combined after review via TLC on EMFplates using methylene chloride, methanol (7:3) as the eluent and SWUVfor visualization. The combined fractions were concentrated to drynessunder vacuum and the residue subjected to reverse phase purification wasrepeated as previously described. The desired fractions wereconcentrated to dryness under reduced pressure and the residue dissolvedin ethanol. Ethyl acetate was added to the cloud point, then heptaneadded to complete the precipitation. 0.55 g of the product,9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,dihydrate, dipotassium salt, was isolated as a yellow solid. The carbonanalysis correlated with a hydrated structure C₂₃H₂₈O₇K₂.1.75 H₂O:Theory C, 52.50 vs 55.85 for anhydrous form; Found C, 52.49. After TLCon EMF plates with methylene chloride, methanol, water (6:3:0.5, v/v) asthe eluent and visualization via SWUV, an R_(f) of 0.29 was observed.

EXAMPLE 47F Preparation of9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,disodium salt

About 5 mg (0.01 mmol) of epoxymexrenone prepared in the manner ofExample 43 was suspended in about 200 μL of methanol in a 4 mL vial anddiluted with about 200 μL of 2.5 N NaOH. The resulting mixture wasyellow and homogeneous. The mixture was then heated in an oil bath at70° C. After 10 minutes a 1 μm sample from the mixture was analyzed byHPLC (Zorbax SB-C8 150×4.6 nn, 2 mL/minute, gradient=35:65 (v/v) A:B,A=acetonitrile/methanol (1:1), B=water/0.1% trifluoroacetic acid,detection at 210 nm) showed two materials at 4.86 and 2.93 minuteretention times consistent with the hydroxyacid (open lactone) and theopen lactone 7-carboxylic acid, respectively. After 30 minutes a second(0.05 mL) sample was removed and acidified with 0.05 mL of 3 N HClfollowed by neutralization with about 0.5 mL of sodium bicarbonate. HPLCanalysis as above showed the expected ring-closed steroids withretention times of 6.59 and 10.71 minutes. The ratio of 7-methylhydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone (10.71 minutes) to the corresponding 7-carboxylic acid was7:89.

Selective hydrolysis of the lactone was possible under mild conditions.A second 4 mL vial was prepared as above but was not heated. The mixturewas sonicated for 5 minutes. A 0.05 mL sample was diluted in 0.5 mL of a1:1 (v/v) mixture of methanol/acetonitrile and was analyzed by HPLCwithout prior acidification. The resulting open lactone carboxylic acid7-ester had a retention time of 4.85 minutes as observed above and wasuncontaminated by the 7-carboxylic acid.

EXAMPLE 47G Isolation of 7-methyl hydrogen9α,11β,17-trihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone

and

7-methyl hydrogen12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone

7-methyl hydrogen9α,11β,17-trihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone and 7-methyl hydrogen12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone were isolated after preparative liquid chromatography of the2-butanone mother liquor recovered from the epoxidation of the enesteras described in example 26 (trichloroacetonitrile protocol). Thus, thefirst crystallization was carried out using 2-butanone as indicated. Therecrystallization, however, utilized 2-butanone (10 vols per g) in placeof acetone. A 2.8 g residue was obtained in this manner and was purifiedvia reverse phase preparative HPLC. Cromasil C8 (10μ) was used as thestationary phase with a mobile phase composed of milliQ water andacetonitrile in a ratio of 70:30 (v/v). Crystallization was observed inone of the enriched fractions. The solid (46.7 mg) was isolated andidentified as 7-methyl hydrogen9α,11β,17-trihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone. The mother liquor was evaporated to dryness under reducedpressure and the residue (123 mg) identified as 7-methyl hydrogen12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone.

7-methyl hydrogen9α,11β,17-trihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone:

MS M+432, calculated for C₂₄H₃₂O₅ (432.51).

¹H NMR (CDCl₃) ppm 1.23 (s,3H), 1.54 (s,3H), 3.00 (m,1H), 3.14 (m,1H),3.74 (s,3H), 5.14 (s,1H, slowly exchangeable), 5.79 (s,1H).

¹³C NMR (CDCl₃) ppm 16.8, 22.7, 24.8, 29.0, 29.3, 32.1, 34.1, 34.7,35.2, 35.7, 36.8, 40.7, 43.0, 45.0, 45.9, 52.9, 72.8, 77.4, 95.9, 127.4,163.7, 176.7, 177.3, 199.4.

7-methyl hydrogen12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-diene-7α,21-dicarboxylate,γ-lactone:

MS M+ 441, calculated for C₂₄H₃₀O₆ (414.50).

¹H NMR (CDCl₃) ppm 0.87 (s,1H), 1.40 (s,1H), 3.05 (m,1H), 3.63 (s,3H),3.99 (m,1H), 5.72 (s,1H), 5.96 (m,1H).

¹³C NMR (CDCl₃) ppm 14.8, 24.0, 26.1, 29.7, 33.6, 33,8, 34.0, 36.3,37.0, 37.4, 40.7, 40.9, 43.8, 48.1, 51.9, 69.1, 95.5, 122.7, 126.3,145.9, 164.5, 173.2, 177.6, 198.2.

EXAMPLE 47H Preparation of 7-methyl hydrogen9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone

and

7-methyl hydrogen6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone

7-methyl hydrogen9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone was prepared according to the method of R. M. Weier and L. M.Hofmann (J. Med Chem 1977, 1304) which is incorporated by reference. 148g (357 mmols) of 7-methyl hydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone prepared in the manner of Example 43 were combined with 311 mLof absolute ethanol and 155 mL (932 mmols) of triethylorthoformate. Theslurry was stirred at room temperature and 10.4 g (54.7 mmols) oftoluene sulfonic acid (monohydrate) added as a catalyst. Stirring wascontinued for 30 minutes and the reaction quenched with the addition of41.4 g (505 mmols) of powdered sodium acetate and 20.7 mL (256 mmols) ofpyridine. Solids (70.2 g) were removed by filtration and the filtrateconcentrated to dryness under vacuum. The residue was digested with 300mL of ethyl acetate and 9.8 g of solids were removed via filtration.

The filtrate was concentrated to dryness and the residue digested with100 mL of methanol containing 2 mL of pyridine. 29.7 g of solids wereremoved via filtration. Additional precipitation was observed in thefiltrate. Therefore, the filtrate was refiltered to remove an additional21.9 g of solids. The filtrate was concentrated to dryness and theresidue digested with 50 mL of methanol containing 1 mL of pyridine.33.8 g of solids were isolated via filtration. Qualitative HPLCindicated that this last portion of solids was sufficiently pure (90area percent of 7-methyl hydrogen9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone) for use in the next step of the reaction.

7-methyl hydrogen9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone:

¹H NMR (CDCl₃) ppm 1.02 (s,3H), 1.27 (s,3H), 1.30 (t,3H), 3.12 (m,1H),3.28 (m,1H), 3.66 (s,3H), 3.78 (m,2H), 5.20 (s,1H), 5.29 (d,1H).

An 8 g portion of the enol ether (7-methyl hydrogen9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone) (18 mmols) prepared in the prior step was dissolved in 120 mLof 1,4-dioxane. The solution was combined with a mixture of 6.8 g of 53%m-chloroperbenzoic acid (20.9 mmols), 18.5 mL of 1.0 N sodium hydroxide(18.5 mmols) and 46 mL of dioxane/water (9:1). The temperature wasmaintained at −3° C. and the mixture stirred for two hours. Thetemperature was raised to 25° C. and stirring continued for anothertwenty hours. The mixture was combined with 400 mL of cold water (10°C.) and 23.5 mL of 1.0 N sodium hydroxide (23.5 mmols). The mixture wasextracted four times with 100 mL portions of methylene chloride eachtime. The combined methylene chloride portions were dried over magnesiumsulfate and the supernatant solvent removed under vacuum distillation.The 13.9 g residue was triturated with 50 mL of ethyl ether to give 2.9g of a white solid. A 2.4 g portion of the solid was chromatographedover 100 g of Merck silica gel (60μ). After an initial washing with 1 Lof 1:1 ethyl acetate/heptane, the product was eluted with a 7:3 ratio ofethyl acetate/heptane. Enriched fractions were combined on the basis ofTLC evaluation (EMF plates; ethyl acetate/heptane 7:3 (v/v) eluent; SWUVvisualization). Thus, 0.85 g of enriched material was obtained andrecrystallized from 10 mL of isopropanol to give 0.7 g of 7-methylhydrogen6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone. The more contaminated fractions were combined and 0.87 g ofcrude 7-methyl hydrogen6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone obtained. This material was chromatographed over 67.8 g ofMerck silica gel (40-63μ). An additional 0.69 g of product was recoveredwith toluene containing 0.5 to 2.5% methanol.

7-methyl hydrogen6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone:

Theory: C, 66.96 and H, 7.02: Found: C, 66.68 and H, 7.16.

¹H NMR (CDCl₃) ppm 1.06 (s,3H), 1.36 (dm,1H), 1.63 (s,3H), 2.92 (m,1H),3.02 (dd,1H), 3.12 (d,1H), 3.64 (s,3H), 4.61 (d,1H), 5.96 (s,1H).

¹³C NMR (CDCl₃) ppm 16.17, 21.32, 21.79, 24.36, 27.99, 28.94, 30.86,31.09, 32.75, 33.19, 34.92, 36.77, 39.16, 43.98, 47.74, 51.56, 51.66,65.36, 72.23, 94.79, 165.10, 171.36, 176.41, 199.59.

EXAMPLE 47I Preparation of 7-methyl hydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7β,21-dicarboxylate,γ-lactone

To 2 g (4.8 mmol) of 7-methyl hydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone prepared in the manner of Example 43 was added 3.3 mL (14.4mmol) of 25% sodium methoxide in methanol. The resulting yellowsuspension was heated to 50° C. The solid did not dissolve. To themixture was added 3.3 ml of methanol (Aldrich anhydrous). The mixturewas heated to reflux conditions (65° C.) and became homogeneous. After30 minutes a solid precipitate prevented stirring.

About 25 ml of anhydrous methanol was added and the mixture wastransferred to a 100 mL flask. The mixture was heated at refluxconditions for 16 hours at which time the mixture was dark andhomogeneous. The mixture was cooled to 25° C. and 70 ml of 3N HCl wasadded (exothermic). Several grams of ice were added to cool the mixtureand the solution was extracted with two successive 25 mL portions ofmethylene chloride. The dark solution was dried over sodium sulfate andfiltered through a 2.5 cm pad of silica gel (E. Merck, 70-230 mesh 60 Åpore size). The silica was eluted with 100 mL of methylene chloride. Theeluted methylene chloride was then concentrated in vacuo to give 1 g ofa brown foam which crystallized upon addition of ethyl acetate. Thesilica pad was eluted a second time with 100 ml of 10% ethylacetate/methylene chloride and the eluted solution was concentrated togive 650 mg of a brown foam.

Thin layer chromatography (E. Merck 60 F-254 silica gel 0.25 mm,toluene/ethyl acetate (1:1, v/v)) revealed the presence of 7-methylhydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone and 7-methyl hydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7β,21-dicarboxylate,γ-lactone in both samples, although very little of the 7α-carboxy epimerwas present in the first sample. The first sample was triturated withhot ethyl acetate (77° C.) and allowed to cool to 25° C. The mixturethen was filtered to give 400 mg of an off-white solid, mp 254-258° C.H, ¹³C and ¹³C-APT were consistent with the assigned structure. A smallamount of ethyl acetate remained in the sample but no starting materialwas evident by HPLC (Zorbax SB-C8 150×4.6 nn, 2 mL/minute, isocratic40:60 (v/v) A:B, A=acetonitrile/methanol (1:1), B=water/0.1%trifluoroacetic acid, detection at 210 nm) (HPLC showed 98.6 areapercent), and by TLC (toluene-ethyl acetate 1:1, v/v).

FAB-MS confirmed a molecular weight of 414 with M₊H at 415.2.

¹H NMR (400 MHz, deuterochloroform) δ 0.95 (s,3H), 1.50 (s,3H), 1.45(m,3H), 1.55-2.7 (m,15H), 2.85 (t,J=13,1H), 3.25 (d,J=6,1H), 3.65(s,3H), 5.78 (s,1H).

EXAMPLE 47J Preparation of9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid,γ-lactone

To 774 mg (1.82 mml) of 7-methyl hydrogen9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylate,γ-lactone prepared in the manner of Example 43 and suspended in 3 ml ofacetonitrile was added 3 mL (7.5 mmol, 2.0 equivalents) of 2.5 M sodiumhydroxide. The mixture became yellow and after 10 minutes washomogeneous.

To monitor the progress of the reaction, aliquots (0.1 mL) of themixture were quenched in 0.01 mL of 3M sulfuric acid and extracted in a4 mL glass vial with ethyl acetate (0.2 ml). The phases were separatedby removal of the lower aqueous phase with a pipette. The organic phasewas stripped and the residue analyzed by HPLC using the method describedin Example 47H. After 50 minutes at 25° C. there was little change inthe composition of the mixture.

The mixture was heated to reflux conditions (about 90° C.) for 50minutes. HPLC analysis of the mixture showed 6 area percent of thestarting material remained. The mixture was stirred at 25° C. for 65hours. Acidification, extraction and HPLC analysis of an aliquot asdescribed above confirmed that no starting material remained.

The mixture was made strongly acidic by addition of about 4 mL of 3Msulfuric acid and was extracted with two portions (about 10 mL) ofmethylene chloride. The organic phases were combined and dried oversodium sulfate. Concentration on a rotary evaporator yielded 780 g of asolid. The solid was recrystallized from dimethyl formamide/methanol togive 503 mg (67%) of a tan crystalline solid. The sample melted with gasevolution near 260° C. when heated rapidly. The sample slowly darkenedbut remained solid when slowly heated to 285° C.

¹H NMR (dimethylsulfoxide d-6, 400 MHz) δ 0.85 (s,3H), 1.4 (s,3H),1.3-2.9 (m,19H), 3.15 (m,1H), 5.55 (s,1H), 11.8 (br,1H).

EXAMPLE 47K Scheme 1: Step 3D: Method I: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

A 0.2 M solution of the enester of Formula IIA in methylene chloride wascombined with 2 equivalents of dipotassium phosphate dissolved in anequal weight of water (50% w/w aqueous solution), 3 equivalents ofchlorodifluoroacetamide and 22 equivalents of hydrogen peroxide (addedas a 30% aqueous solution). The mixture was stirred at 25° C. for 23hours. The reaction was diluted with an amount of water equal to thehydrogen peroxide charge and the methylene chloride separated. Themethylene chloride portion was washed one time with a 3% sodium sulfitesolution (volume equal to 1.75 times the hydrogen peroxide charge). Themethylene chloride portion was separated and dried over sodium sulfate.The solution was concentrated under atmospheric distillation until ahead temperature of 70° C. was achieved. The residue was evaluated viaHPLC, ₁H and ¹³C NMR (CDCl₃) The yield of epoxymexrenone was determinedto be 54.2 area % by HPLC.

EXAMPLE 47L Scheme 1: Step 3D: Method J: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

The procedure of Example 47K was repeated using heptafluorobutyramide(CF₃CF₂CF₂CONH₂) instead of chlorodifluoroacetamide. The yield ofepoxymexrenone was determined to be 58.4 area % by HPLC.

EXAMPLE 48 Epoxidation of Enester of Formula IIA Using Toluene Scheme 1:Step 3D: Method K: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone

The enester of Formula IIA was converted to epoxymexrenone in the methodgenerally described in Example 46 except that toluene was used as thesolvent. The materials charged to the reactor included enester (2.7 g)trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g),hydrogen peroxide (17.0 g) and toluene (50 ml). The reaction was allowedto exotherm to 28° C. and was complete in 4 hours. The resulting threephase mixture was cooled to 15° C., filtered, washed with water anddried in vacuo to yield 2.5 g of product.

EXAMPLE 49 Scheme 4: Method A: Epoxidation of 9,11-Dienone

A compound designated XVIIA (compound XVII wherein —A—A— and —B—B— areboth —CH₂—CH₂—) (40.67 g) was dissolved in methylene chloride (250 mL)in a one liter 3 necked flask and cooled by ice salt mixture externally.Dipotassium phosphate (22.5 g), and trichloroacetonitrile (83.5 g) wereadded and mixture cooled to 2° C. after which 30% Hydrogen peroxide (200g) was slowly added over a period of 1 hour. The reaction mixture wasstirred at 12° for 8 hours and 14 hours at room temperature. A drop ofthe organic layer was taken and checked for any starting enone and wasfound to be <0.5%. Water (400 mL) was added, stirred for 15 min. andlayers separated. The organic layer was washed successively with 200 mLof potassium iodide (10%), 200 mL of sodium thiosulfate (10%) and 100 mLof saturated sodium bicarbonate solution separating layers each time.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to yield crude epoxide (41 g). The product crystallizedfrom ethyl acetate:methylene chloride to give 14.9 g of pure material.

EXAMPLE 50 Scheme 4: Method B: Epoxidation of Compound XVIIA Usingm-chloroperbenzoic Acid

Compound XVIIA (18.0 g) was dissolved in 250 mL of methylene chlorideand cooled to 10° C. Under stirring solid m-chloroperbenzoic acid,(50-60% pure, 21.86 g) was added during 15 min. No rise in temperaturewas observed. The reaction mixture was stirred for 3 hours and checkedfor the presence of the dienone. The reaction mixture was treatedsuccessively with sodium sulfite solution (10%), sodium hydroxidesolution (0.5N), hydrochloric acid solution (5%) and finally with 50 mLof saturated brine solution. After drying with anhydrous magnesiumsulfate and evaporation, 17.64 g of the epoxide resulted and was useddirectly in the next step. The product was found to containBaeyer-Villiger oxidation product that had to be removed by triturationfrom ethyl acetate followed by crystallization from methylene chloride.On a 500 g scale, the precipitated m-chlorobenzoic acid was filteredfollowed by the usual work up.

EXAMPLE 51 Scheme 4: Method C: Epoxidation of Compound XVIIA UsingTrichloroacetamide

Compound XVIIA (2 g) was dissolved in 25 mL of methylene chloride.Trichloroacetamide (2 g), dipotassium phosphate (2 g) were added. Understirring at room temperature 30% hydrogen peroxide (10 mL) was added andstirring continued for 18 hours to yield the epoxide (1.63 g).Baeyer-Villiger product was not formed.

EXAMPLE 52

Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 eq.) was charged to a2000 mL flask and slurried with dimethylsulfoxide (750.0 mL) at ambienttemperature. A trienone corresponding to Formula XX (wherein R³ is H and—A—A— and —B—B— are each —CH₂—CH₂—) (100.00 g; 335.01 mmol; 1.00 eq.)was charged to the flask together with THF (956.0 mL).Trimethylsulfonium methylsulfate (126.14 g; 670.02 mmol; 2.00 eq.) wascharged to the flask and the resulting mixture heated at reflux, 80 to85° C. for 1 hr. Conversion to the 17-spirooxymethylene was checked byHPLC. THF approximately 1 L was stripped from the reaction mixture undervacuum after which water (460 mL) was charged over a 30 min. periodwhile the reaction mixture was cooled to 15° C. The resulting mixturewas filtered and the solid oxirane product washed twice with 200 mLaliquots of water. The product was observed to be highly crystalline andfiltration was readily carried out. The product was thereafter driedunder vacuum at 40° C. 104.6 g of the 3-methyl enol etherΔ-5,6,9,11,-17-oxirane steroid product was isolated.

EXAMPLE 53

Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) was charged to a dry500 mL reactor under a nitrogen blanket. Ethanol (270.9 mL) was chargedto the reactor and the sodium methoxide slurried in the ethanol. Diethylmalonate (103.90 g; 648.68 mmol; 2.00 eq.) was charged to the slurryafter which the oxirane steroid prepared in the manner described inExample 52 (104.60 g; 324.34 mmol; 1.00 eq.) was added and the resultingmixture heated to reflux, i.e., 80 to 85° C. Heating was continued for 4hrs. after which completion of the reaction was checked by HPLC. Water(337.86 mL) was charged to the reaction mixture over a 30 min. periodwhile the mixture was being cooled to 15° C. Stirring was continued for30 min. and then the reaction slurry filtered producing a filter cakecomprising a fine amorphous powder. The filter cake was washed twicewith water (200 mL each) and thereafter dried at ambient temperatureunder vacuum. 133.8 g of the 3-methylenolether-Δ5,6,9,11,-17-spirolactone-21-ethoxycarbonyl intermediate wasisolated.

EXAMPLE 54

The 3-methyl enolether-Δ5,6,9,11,-17-spirolactone-21-ethoxycarbonylintermediate (Formula XVIII where R³ is H and —A—A— and —B—B— are each—CH₂—CH₂—; 133.80 g; 313.68 mmol; 1.00 eq., as produced in Example 53,was charged to the reactor together with sodium chloride (27.50 g;470.52 mmol; 1.50 eq.) dimethyl formamide (709 mL) and water (5 mL) werecharged to a 2000 mL reactor under agitation. The resulting mixture washeated to reflux, 138 to 142° C. for 3 hrs. after which the reactionmixture was checked for completion of the reaction by HPLC. Water wasthereafter added to the mixture over a 30 min. period while the mixturewas being cooled to 15° C. Agitation was continued for 30 min. afterwhich the reaction slurry was filtered recovering amorphous solidreaction product as a filter cake. The filter cake was washed twice (200mL aliquots of water) after which it was dried. The product3-methylenolether-17-spirolactone was dried yielding 91.6 g (82.3%yield; 96 area % assay).

EXAMPLE 55

The enol ether produced in accordance with Example 54 (91.60 g; 258.36mmol; 1.00 eq.) ethanol (250 mL) acetic acid (250 mL) and water (250 mL)were charged to a 2000 mL reactor and the resulting slurry heated toreflux for 2 hrs. Water (600 mL) was charged over a 30 min. period whilethe reaction mixture was being cooled to 15° C. The reaction slurry wasthereafter filtered and the filter cake washed twice with water (200 mLaliquots). The filter cake was then dried; 84.4 g of product 3-ketoΔ4,5,9,11,-17-spirolactone was isolated (compound of Formula XVII whereR³ is H and —A—A— and —B—B— are —CH₂—CH₂—; 95.9% yield).

EXAMPLE 56

Compound XVIIA (1 kg; 2.81 moles) was charged together with carbontetrachloride (3.2 L) to a 22 L 4-neck flask. N-bromo-succinamide (538g) was added to the mixture followed by acetonitrile (3.2 L). Theresulting mixture was heated to reflux and maintained at the 68° C.reflux temperature for approximately 3 hrs. producing a clear orangesolution. After 5 hrs. of heating, the solution turned dark. After 6hrs. the heat was removed and the reaction mixture was sampled. Thesolvent was stripped under vacuum and ethyl acetate (6 L) added to theresidue in the bottom of the still. The resultant mixture was stirredafter which a 5% sodium bicarbonate solution (4 L) was added and themixture stirred for 15 min. after which the phases were allowed tosettle. The aqueous layer was removed and saturated brine solution (4 L)introduced into the mixture which was then stirred for 15 min. Thephases were again separated and the organic layer stripped under vacuumproducing a thick slurry. Dimethylformamide (4 L) was then added andstripping continued to a pot temperature of 55° C. The still bottomswere allowed to stand overnight and DABCO (330 g) and lithium bromide(243 g) added. The mixture was then heated to 70° C. After one andone-half hrs. heating, a liquid chromatography sample was taken andafter 3.50 hrs. heating, additional DABCO (40 g) was added. After 4.5hrs. heating, water (4 L) was introduced and the resulting mixture wascooled to 15° C. The slurry was filtered and the cake washed with water(3 L) and dried on the filter overnight. The wet cake (978 g) wascharged back into the 22 L flask and dimethylformamide (7 L) added. Themixture thus produced was heated to 105° C. at which point the cake hadbeen entirely taken up into solution. The heat was removed and themixture in the flask was stirred and cooled. Ice water was applied tothe reactor jacket and the mixture within the reactor cooled to 14° C.and held for two hours. The resulting slurry was filtered and washedtwice with 2.5 L aliquots of water. The filter cake was dried undervacuum overnight. A light brown solid product 510 g was obtained.

EXAMPLE 57

To a 2 L 4-neck flask were charged: 9,11-epoxy canrenone as produced inExample 56 (100.00 g; 282.1 mmol; 1.00 eq.), dimethylformamide (650.0mL), lithium chloride (30.00 g; 707.7 mmol; 2.51 eq.), and acetonecyanohydrin (72.04 g; 77.3 mL; 846.4 mmol; 3.00 eq.). The resultingsuspension was mechanically stirred and treated with tetramethylguanidine (45.49 g; 49.6 mL; 395.0 mmol; 1.40 eq.). The system was thenfiltered with a water cooled condenser and a dry ice condenser (filledwith dry ice in acetone) to prevent escape of HCN. The vent line fromthe dry ice condenser passed into a scrubber filled with a large excessof chlorine bleach. The mixture was heated to 80° C.

After 18 hrs., a dark reddish-brown solution was obtained which wascooled to room temperature with stirring. During the cooling process,nitrogen was sparged into the solution to remove residual HCN with thevent line being passed into bleach in the scrubber. After two hrs. thesolution was treated with acetic acid (72 g) and stirred for 30 min. Thecrude mixture was then poured into ice water (2 L) with stirring. Thestirred suspension was further treated with 10% aqueous HCl (400 mL) andstirred for 1 hr. Then the mixture was filtered to give a dark brick-redsolid (73 g). The filtrate was placed in a 4 L separatory funnel andextracted with methylene chloride (3×800 mL); and the organic layerswere combined and back extracted with water (2×2 L). The methylenechloride solution was concentrated in vacuo to give 61 g of a dark redoil.

After the aqueous wash fractions were allowed to sit overnight, aconsiderable precipitate developed. This precipitate was collected byfiltration and determined to be pure product enamine (14.8 g).

After drying the original red solid (73 g) was analyzed by HPLC and itwas determined that the major component was the 9,11-epoxyenamine. HPLCfurther showed that enamine was the major component of the red oilobtained from methylene chloride workup. Calculated molar yield ofenamine was 46%.

EXAMPLE 58

9,11-epoxyenamine (4.600 g; 0.011261 mol; 1.00 eq.) as prepared inaccordance with Example 57 was introduced into a 1000 mL round bottomflask. Methanol (300 mL) and 0.5% by weight aqueous HCl (192 mL) wereadded to the mixture which was thereafter refluxed for 17 hrs. Methanolwas thereafter removed under vacuum reducing the amount of material inthe still pot to 50 mL and causing a white precipitate to be formed.Water (100 mL) was added to the slurry which was thereafter filteredproducing a white solid cake which was washed three times with water.Yield of solid 9,11-epoxydiketone product was 3.747 g (81.3%).

EXAMPLE 59

The epoxydiketone prepared in accordance with Example 58 (200 mg; 0.49mmol) was suspended in methanol (3 mL) and1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) added to the mixture. Uponheating under reflux for 24 hrs. the mixture became homogeneous. It wasthen concentrated to dryness at 30° C. on a rotary evaporator and theresidue partitioned between methylene chloride and 3.0 N HCl.Concentration of the organic phase yielded a yellow solid (193 mg) whichwas determined to be 22% by weight epoxy mexrenone. The yield was 20%.

EXAMPLE 60

To 100 mg of diketone (prepared in accordance with Example 58) suspendedin 1.5 mL of methanol was added 10 microliters (0.18 eq) of a 25% (w/w)solution of sodium methoxide in methanol. The solution was heated toreflux. After 30 min. no diketone remained and the 5-cyanoester waspresent. To the mixture was added 46 microliters of 25% (w/w) sodiummethanol solution in methanol. The mixture was heated at reflux for 23hours at which time the major product was epoxymexrenone as judged byHPLC.

EXAMPLE 61

To 2 g of diketone (prepared in accordance with Example 58) suspended in30 ml of dry methanol was added 0.34 mL of triethylamine. The suspensionwas heated at reflux for 4.5 hours. The mixture was stirred at 25° C.for 16 hours. The resulting suspension was filtered to give 1.3 g of the5-cyanoester as a white solid.

To 6.6 g of the diketone suspended in 80 mL of methanol was added 2.8 mLof triethylamine. The mixture was heated at reflux for 4 hours and wasstirred at 25 rpm for 88 hours during which time the productcrystallized from solution. Filtration followed by a methanol wash gave5.8 g of the cyanoester as a white powder. The material wasrecrystallized from chloroform/methanol to give 3.1 g of crystallinematerial which was homogeneous by HPLC.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above compositions and processeswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

The Novel Compounds

The present invention is further directed to additional polycyclicorganic moieties useful as chromatographic markers in the preparation ofsteroid compounds having favorable biological activity such asspironolactone or epoxymexrenone.

In brief, it has been discovered that certain compounds comprising asubstituted or unsubstituted steroid nucleus and a substituted orunsubstituted carbocyclic ring fused to the 13,17 position of thesteroid nucleus can used as internal or chromatographic markers in thepreparation of steroids such as spironolactone and epoxymexrenone. Inparticular, the compound methyl 2,3,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3aβ,11aβ-dimethyl-1,9-dioxo-1H-pentaleno[1,6a-a]phenanthrene-6α-carboxylate:

is useful as a chromatographic marker. One of the novel features of thiscompound and the related compounds of this invention is the fusedcarbocyclic ring attached to the D ring of the steroid nucleus.Spironolactone and epoxymexrenone lack this feature and instead possessa 20-spirolactone ring.

As used herein, the steroid nucleus of the compound corresponds to thefollowing structure:

This structure reflects the conventional numbering and ring designationfor steroid compounds. The steroid nucleus may be saturated,unsaturated, substituted or unsubstituted. Preferably, it contains atleast one to four unsaturated bonds. More preferably, the A, C and Drings each contain at least one unsaturated bond. The steroid nucleusalso may be substituted as more specifically discussed below.Preferably, the nucleus is substituted with at least a C7 ester group.

As used herein, the carbocyclic ring fused to the steroid nucleuscorresponds to a four, five or six carbon cyclic skeleton. It may besaturated, unsaturated, substituted or unsubstituted. Preferably, it issaturated or has one double bond, and it is substituted with a hydroxyor keto group. In addition, the carbocyclic ring preferably has theα-orientation relative to the steroid nucleus.

In a preferred embodiment, the carbocyclic ring comprises a five carboncyclic skeleton and has the α-orientation, and the compound is selectedfrom the group consisting of those compounds corresponding to thefollowing formulae:

wherein

—A—A— represents the group —CR¹⁰²R^(102a)—CR¹⁰³R^(103a)— or—CR¹⁰²═CR¹⁰³—, wherein —CR¹⁰²R^(102a)— and —CR¹⁰²═ correspond to the C2carbon, and —CR¹⁰³R^(103a)— and ═CR¹⁰³— correspond to the C3 carbon;

—D—D— represents the group —CHR¹⁰⁴—CH— or —CR¹⁰⁴═C—;

—E—E— represents the group —CH₂—CR¹¹⁰— or —CH═C—;

—A—E— represents the group —CR¹⁰²R^(102a)—CH₂— or —CR¹⁰²═CH, wherein—CR¹⁰²R^(102a)— and —CR¹⁰²═ correspond to the C2 carbon and —CH₂— and═CH— correspond to the C1 carbon;

—G—G— represents the group —CR¹⁰⁶R^(106a)—CHR¹⁰⁷— or —CR¹⁰⁶═CR¹⁰⁷—,wherein —CR¹⁰⁶R^(106a)— and —CR¹⁰⁶═ correspond to the C6 carbon and—CHR¹⁰⁷— and ═CR¹⁰⁷— correspond to the C7 carbon;

—J—J— represents the group —CR¹⁰⁸—CR¹⁰⁹— or —C═C—, wherein —CR¹⁰⁸—corresponds to the C8 carbon and —CR¹⁰⁹— corresponds to the C9 carbon;

—L—L— represents the group —CR¹¹¹R^(111a)—CH₂— or —CR¹¹¹═CH—, wherein—CR¹¹¹R^(111a)— and —CR¹¹¹═ correspond to the C11 carbon and —CHR²— and═CH— correspond to the C12 carbon;

—J—L— represents the group —CR¹⁰⁹—CR¹¹¹R^(111a)— or —C═CR¹¹¹—, wherein—CR¹⁰⁹— and —C═ correspond to the C9 carbon, and —CR¹¹¹R^(111a)— and—CR¹¹¹— correspond to the C11 carbon;

—M—M— represents the group —CR¹¹⁴—CH₂— or —C═CH—, wherein —CR¹¹⁴— and—C═ correspond to the C14 carbon, and —CH₂— and —C═H— correspond to theC15 carbon;

—J—M— represents the group —CR¹⁰⁸—CR¹¹⁴— or —C═C—, wherein —CR¹⁰⁸—corresponds to the C8 carbon and —CR¹¹⁴— corresponds to the C14 carbon;

—Q—Q— represents the group —CR¹²⁰R^(120a)—CR¹¹⁹R^(119a)— or—CR¹²⁰═CR¹¹⁹—, wherein —CR¹²⁰R^(120a)— and —CR¹²⁰═CR¹¹⁹— correspond tothe C20 carbon, and —CR¹¹⁹R^(119a)— and ═CR¹¹⁹— correspond to the C19carbon;

—Q— T—represents the group —CR¹¹⁹R^(119a)—CHR¹¹⁸— or —CR¹¹⁹═CR¹¹⁸—,wherein —CR¹¹⁹R^(119a)— and —CR¹¹⁹═ correspond to the C19 carbon, and—CHR¹¹⁸— and ═CR¹¹⁸— correspond to the C18 carbon;

R¹⁰² is hydrogen, alkyl, alkenyl or alkynyl;

R^(102a) is hydrogen; or represents a bond between the C2 carbon atomand the C3 carbon atom when —A—A— represents the group —CR¹⁰²═CR¹⁰³— and—A—E— represents the group —CR¹⁰²R^(102a)—CH₂—; or represents a bondbetween the C1 carbon atom and the C2 carbon atom when —A—E— representsthe group —CR¹⁰²═CH— and —A—A— represents the group—CR¹⁰²R^(102a)—CR¹⁰³R^(103a)—;

R¹⁰³ is hydrogen, hydroxy, protected hydroxy, R¹³⁰O—, R¹³⁰C(O)O—,R¹³⁰OC(O)O—, or together with R^(103a) forms an oxo; provided that —A—A—is —CR¹⁰²R^(102a)—CR¹⁰³R^(103a)— when R¹⁰³ together with R^(103a) forman oxo;

R^(103a) is hydrogen or together with R¹⁰³ forms an oxo; provided that—A—A— is —CR¹⁰²R^(102a)—CR¹⁰³R^(103a)— when R^(103a) together with R¹⁰³form an oxo;

R¹⁰⁴ is hydrogen, alkyl, alkenyl or alkynyl;

R¹⁰⁶ is hydrogen, hydroxy or protected hydroxy, or together withR^(106a) forms an oxo, or together with R^(106a) and the carbon atom towhich they are attached form a cyclopropyl, cyclobutyl or cyclopentylring; provided that —G—G— is —CR¹⁰⁶R^(106a)—CR¹⁰⁷R^(107a)— when R¹⁰⁶together with R^(106a) form an oxo;

R^(106a) is hydrogen, hydroxy or protected hydroxy, or together withR^(106a) forms an oxo, or together with R¹⁰⁶ and the carbon atom towhich they are attached form a cyclopropyl, cyclobutyl or cyclopentylring, or R^(106a) together with R¹⁰⁷ and the carbon atoms to which theyare attached form a cyclopropyl, cyclobutyl or cyclopentyl ring;

R¹⁰⁷ is hydrogen; hydroxycarbonyl; lower alkyl, alkenyl, alkynyl, aryl,heteroaryl or aralkyl; haloalkyl; hydroxyalkyl; alkoxyalkyl; loweralkanoyl, alkenoyl, alkynoyl, aryloyl, heteroaryloyl or aralkanoyl;lower alkoxycarbonyl, alkenoxycarbonyl, alkynoxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl or aralkoxycarbonyl; loweralkanoylthio, alkenoylthio, alkynoylthio, aryloylthio, heteraroylthio oraralkanoylthio; lower alkylthio, alkenylthio, alkynylthio, arylthio,heteroarylthio or aralkylthio; carbamyl; alkoxycarbonylamino; or cyano;or

R¹⁰⁷ together with R^(106a) and the carbon atoms to which they areattached form a cyclopropyl, cyclobutyl or cyclopentyl ring; or

R¹⁰⁷ and R¹¹⁴ together with the C7, C8 and C14 carbon atoms form aγ-lactone;

R¹⁰⁸ is hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl,R¹⁴⁰O—, R¹⁴⁰C(O)O—, or R¹⁴⁰OC(O)O—; or represents a bond between the C8carbon atom and the C9 carbon atom when —J—J— represents the group —C═C—and —J—M— represents the group —CR¹⁰⁸—CR¹¹⁴—; or represents a bondbetween the C8 carbon atom and the C14 carbon atom when —J—M— representsthe group —C═C— and —J—J— represents the group —CR¹⁰⁸—CR¹¹⁴—;

R¹⁰⁹ is hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl,R¹⁵⁰O—, R¹⁵⁰C(O)O—, or R¹⁵⁰OC(O)O—; or represents a bond between the C9carbon atom and the C11 carbon atom when —J—L— represents the group—C═CR¹¹¹— and —J—J— represents the group —CR¹⁰⁸—CR¹⁰⁹—; or represents abond between the C9 carbon atom and the C8 carbon atom when —J—J—represents the group —C═C— and —J—L— represents the group—CR¹⁰⁹—CR^(111a)—;

R¹¹⁰ is hydrogen or methyl;

R¹¹¹ is hydrogen, hydroxy or protected hydroxy, or together withR^(111a) form an oxo, provided that —J—L— represents the group—CR¹⁰⁹—CR¹¹¹R^(111a)— and —L—L— represents the group —CR¹¹¹R^(111a)—CH₂—when R¹¹¹ together with R^(111a) form an oxo;

R^(111a) is hydrogen, or together with R¹¹¹ form an oxo, provided that—J—L— represents the group —CR¹⁰⁹—CR¹¹¹R^(111a)— and —L—L— representsthe group —CR¹¹¹R^(111a)—CH₂— when R^(111a) together with R¹¹¹ form anoxo; or R^(111a) represents a bond between the C11 carbon atom and theC9 carbon atom when —J—L— represents the group —C═CR¹¹¹— and —L—L—represents the group —CR¹¹¹R^(111a)—CH₂—; or R^(111a) represents a bondbetween the C11 carbon atom and the C12 carbon atom when —L—L—represents the group —CR¹¹¹═CH— and —J—L— represents the group—CR¹⁰⁹R^(109a)—CR¹¹¹R^(111a)—;

R¹¹⁴ is hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl,R¹⁶⁰O—, R¹⁶⁰C(O)O—, or R¹⁶⁰OC(O)O—; or R¹¹⁴ and R¹⁰⁷ together with theC7, C8 and C14 carbons form a γ-lactone; or R¹¹⁴ represents a bondbetween the C14 carbon atom and the C8 carbon atom when —J—M— representsthe group —C═C— and —M—M— represents the group —CR¹¹⁴—CH₂—; or R¹¹⁴represents a bond between the C14 carbon atom and the C15 carbon atomwhen —M—M— represents the group —C═CH— and —J—M— represents the group—CR¹⁰⁸—CR¹¹⁴—;

R¹¹⁸ is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylthio,alkenylthio or cyano;

R^(118a) is hydrogen, or represents a bond between the C18 carbon atomand the C19 carbon atom when —Q—T— represents the group —CR¹¹⁸═CR¹¹⁹—and —Q—Q— represents the group —CR¹¹⁹R^(119a)—CR¹²⁰R^(120a)—;

R¹¹⁹ is hydrogen, alkyl or alkenyl;

R^(119a) is hydrogen, or represents a bond between the C19 carbon atomand the C20 carbon atom when —Q—Q— represents the group —CR¹²⁰═CR¹¹⁹—and —Q—T— represents the group —CR¹¹⁹R^(119a)—CR¹¹⁸R^(118a)—; orrepresents a bond between the C19 carbon atom and the C18 carbon atomwhen —Q—T— represents —CR¹¹⁹═CR¹¹⁸— and —Q—Q— represents the group—CR¹¹⁹R^(119a)—CR¹²⁰R^(120a)—;

R¹²⁰ is hydrogen, hydroxy, protected hydroxy, or together with R^(120a)forms an oxo; provided that —Q—Q— represents the group—CR¹¹⁹R^(119a)—CR¹²⁰R^(120a) when R¹²⁰ together with R^(120a) form anoxo;

R^(120a) is hydrogen or together with R¹²⁰ form an oxo; provided that—Q—Q— represents the group —CR¹¹⁹R^(119a)—CR¹²⁰R^(120a) when R^(120a)together with R¹²⁰ forms an oxo; and

R¹³⁰, R¹⁴⁰, R¹⁵⁰ and R¹⁶⁰ are independently alkyl, alkenyl, alkynyl,aryl or heteroaryl.

More preferably, the compound corresponds to the compound of Formula C-3wherein R¹⁰⁷ is hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl,lower alkoxycarbonyl, lower alkanoylthio, lower alkylthio, carbamyl, orR¹⁰⁷ together with R^(106a) and the carbon atoms to which they areattached form a cyclopropyl ring; R¹⁰⁶ is hydrogen, hydroxy, or togetherwith R^(106a) and the carbon atom to which they are attached form acyclopropyl ring; R^(106a) is hydrogen, hydroxy, or together with R¹⁰⁶and the carbon atom to which they are attached form a cyclopropyl ring;or R^(106a) together with R¹⁰⁷ and the carbon atoms to which they areattached form a cyclopropyl ring; and R¹²⁰ is keto.

The following definitions apply to the discussion relating to the13,17-fused ring compounds disclosed in the present specification:

The term “lower alkyl” means an alkyl radical having from 1 to 6 carbonatoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec.-butyl and tert.-butyl, pentyl and hexyl. The radical may bestraight, branched chain or cyclic and substituted (particularly witharyl), unsubstituted or heterosubstituted.

The term “lower alkanoyl” means a radical preferably derived from astraight-chain alkyl having from 1 to 7 carbon atoms and attached to theparent molecular moiety via a carbonyl group. Especially preferred areformyl and acetyl.

The term “lower alkoxycarbonyl” means a radical preferably derived froma straight-chain alkyl having from 1 to 7 carbon atoms and attached toan oxygen atom, said oxygen atom being attached to the parent molecularmoiety via a carbonyl group. Especially preferred are methoxycarbonyl,ethoxycarbonyl, isopropoxycarbonyl and n-hexyloxycarbonyl.

The term “lower alkenyl” means an alkenyl radical having from 2 to 6carbon atoms, such as ethenyl, propenyl, isopropenyl, butenyl,isobutenyl, sec.-butenyl and tert.-butenyl, pentenyl and hexenyl. Theradical may be straight or branched chain and substituted, unsubstitutedor heterosubstituted. The terms “lower alkenoyl” and “loweralkenoxycarbonyl” are defined in the same manner as “lower alkanoyl” and“lower alkoxycarbonyl”, respectively, except that they are derived fromstraight chain alkenyl instead of straight chain alkyl. Preferably, theoxygen attached to the alkenyl radical of any alkenoxycarbonyl group isseparated from any unsaturated carbon by at least one methylene group.

The term “lower alkynyl” means an alkynyl radical having from 2 to 6carbon atoms, such as ethynyl, propynyl, isopropynyl, butynyl,isobutynyl, sec.-butynyl and tert.-butynyl, pentynyl and hexynyl. Theradical may be straight or branched chain and substituted, unsubstitutedor heterosubstituted. The terms “lower alkynoyl” and “loweralkynoxycarbonyl” are defined in the same manner as “lower alkanoyl” and“lower alkoxycarbonyl”, respectively, except that they are derived fromstraight chain alkynyl instead of straight chain alkyl. Preferably, theoxygen attached to the alkynyl radical of any alkynoxycarbonyl group isseparated from any unsaturated carbon by at least one methylene group.

An “aryl” moiety preferably contains, either alone or with varioussubstituents, from 5 to 15 atoms and includes phenyl.

The term “lower alkylthio” means a radical preferably derived from astraight-chain alkyl having from 1 to 7 carbon atoms and attached to theparent molecular moiety via a sulfur atom. Especially preferred ismethylthio.

The term “lower alkanoylthio” means a radical preferably derived from astraight-chain alkyl having from 1 to 7 carbon atoms and attached to acarbonyl group, said carbonyl group being attached to the parentmolecular moiety via a sulfur atom. Especially preferred is acetylthio.

The terms “lower alkenylthio” and “lower alkenoylthio” are defined inthe same manner as “lower alkylthio” and “lower alkanoylthio”,respectively, except that they are derived from straight chain alkenylinstead of straight chain alkyl. Preferably, the sulfur atom of anyalkenylthio group is separated from any unsaturated carbon by at leastone methylene group.

The terms “lower alkynylthio” and “lower alkynoylthio” are defined inthe same manner as “lower alkylthio” and “lower alkanoylthio”,respectively, except that they are derived from straight chain alkynylinstead of straight chain alkyl. Preferably, the sulfur atom of anyalkynylthio group is separated from any unsaturated carbon by at leastone methylene group.

The term “carbamyl” means an —NH₂ radical attached to the parentmolecular moiety via a carbonyl group. The carbamyl group may bemono-substituted or di-substituted and the substituents may includealkyl, alkenyl, alkynyl and aryl radicals.

The groups defined above may be unsubstituted or additionallysubstituted. Such additional substituents can include alkyl, alkenyl,alkynyl, aryl, heteroaryl, carboxy such as alkoxy, carboxyalkyl, acyl,acyloxy, halo such as chloro or fluoro, haloalkoxy, nitro, amino, amido,and keto. The groups defined above, as well as the additionalsubstituents, also may contain oxygen, sulfur, phosphorus and/ornitrogen.

As used herein, “Me” means methyl; “Et” means ethyl; and “Ac” meansacetyl.

In a still more preferred embodiment, the compound is selected from thegroup consisting of compounds having the following formulae:

wherein R¹⁰⁷, R¹⁰⁶, R^(106a) and R¹²⁰ are as defined above. Preferably,R¹⁰⁷ is hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, loweralkoxycarbonyl, lower alkanoylthio, lower alkylthio, carbamyl, ortogether with R^(106a) and the carbon atoms to which they are attachedform a cyclopropyl ring; R¹⁰⁶ is hydrogen, hydroxy, or together withR^(106a) and the carbon atom to which they are attached form acyclopropyl ring; R^(106a) is hydrogen, hydroxy, or together with R¹⁰⁶and the carbon atom to which they are attached form a cyclopropyl ring;or together with R¹⁰⁷ and the carbon atoms to which they are attachedform a cyclopropyl ring; and R¹²⁰ is keto. Still more preferably, thecompound further corresponds to the compound of Formula C-5.

In an even more preferred embodiment, the compound of Formula C-3 isselected from the group consisting of the following compounds:

In the most preferred embodiment, the compound is methyl2,2,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3αβ,11αβ-dimethyl-1,9-dioxo-1H-pentaleno[1,6a-a]phenantrene-6α-caboxylate:

This compound of Formula C-1 is a particularly desirable chromotographicmarker in the preparation of epoxymexrenone.

The novel compounds discussed herein may also be in the form of theirsalts.

Preparation of the Novel Compounds

In general, the novel compounds described immediately above may beobtained by reacting a steroid having a 20-spiroxane ring and thesteroid nucleus previously described above with a trihalogenatedalkanoic acid. Advantageously, the reagent for the reaction furtherincludes an alkali metal salt of the alkanoic acid utilized. It isparticularly preferred that the reagent for the reaction comprisetrifluoroacetic acid and an alkali metal salt of that acid such aspotassium trifluoroacetate. In addition, a drying agent such astrifluoroacetic anhydride preferably is employed in the reaction toreduce free water present in the acid.

The steroid compounds used as starting materials preferably have thefollowing structural formula:

wherein —A—A—, —D—D—, —E—E—, —A—E—, —G—G—, —J—J—, —L—L—, —J—L—, —M—M—,—J—M—, —Q—Q—, and —Q—T— are as defined above. Such starting materialscan be prepared and/or isolated by processes analogous to thosedisclosed in Scheme 1 of the epoxymexrenone synthesis process previouslydiscussed herein. Alternatively, the starting materials are commerciallyavailable.

The initial concentration of the steroid compound of formula C-3 ispreferably at least about 0.1% by weight of the total reaction mixture,more preferably about 2% to about 20% by weight, and even morepreferably about 5% to about 15% by weight. An excess of thetrihaloalkanoic acid is preferably present. Where trifluoroaceticanhydride is used, it should be present in a proportion of at leastabout 3% by weight of the total reaction mixture, more preferablybetween about 5% to about 25% by weight, most preferably between about10% and about 15% by weight.

In addition, the reaction temperature should exceed room temperature(22° C.). Preferably, the reaction temperature is between about 40° C.and 100° C., more preferably between about 50° C. and 80° C., still morepreferably between about 60° C. and 70° C., and most preferably betweenabout 60° C. and 65° C. Increasing the reaction temperature to about 70°C. or higher increases the amount of the by-product C14 lactone producedin the reaction. The reaction time preferably should be at least about30 minutes, more preferably between about 30 minutes and about 6 hours,still more preferably between about 45 minutes and about 4 hours, andmost preferably between about one hours to two hours. In a preferredembodiment the reaction time is between about one hour to two hours andthe reaction temperature is maintained at about 60° C.

Scheme S-1 below illustrates a particularly preferred embodiment of theprocess:

Fused ring steroids having different substituents at various positionsthroughout the steroid can be prepared as set forth in the reactionschemes below. Those skilled in the art are aware of additionalprocedures and methods not specifically disclosed herein for introducingvarious substituents at different positions of the steroid. The startingmaterial may be either a fused ring steroid or a 20-spiroxane ringsteroid. To simplify the description where the starting material is afused ring steroid, the following reaction schemes employ specificsteroids or groups of steroids as illustrative starting materials. Itshould be understood, however, that other fused ring steroid derivativesor analogs may be produced in the same series of reactions by using adifferent fused ring steroid as the starting material. Similarly, tosimplify the description where the starting material is a 20-spiroxanering steroid, certain specific 20-spiroxane ring steroids are used asthe starting materials. It should be understood, however, that other20-spiroxane ring steroid derivatives or analogs may be produced in thesame series of reactions by using a different 20-spiroxane ring steroidas the starting material. Steroids having a C7 carboxylic acidsubstituent may be prepared by saponification of a steroid having a C7alkoxycarbonyl substituent such as the compound of Formula C-1. Thesaponification reaction may be carried out by treatment of the startingsteroid with a basic reagent such as sodium or potassium hydroxide in asuitable solvent such as methanol, ethanol, isopropanol or the like attemperatures up to the boiling point of the solvent in the presence orabsence of water. As illustrated in Scheme S-2, saponification of thecompound of Formula C-1 yields the carboxylic acid of Formula C-101.

Steroids having C7 carboxylic ester substituents other than methanoatecan be prepared using carboxylic acids such as C-101 as the startingmaterial. Treatment of such carboxylic acids with an alkylating agentsuch as an alkyl halide in the presence of a base (such as sodiumbicarbonate, sodium carbonate, potassium bicarbonate, or triethylamine)in a solvent such as dimethylformamide yields the desired esters.Examples of suitable alkylating agents are ethyl iodide, ethyl bromide,isopropyl iodide, hexyl iodide, benzyl bromide, allyl iodide, and thelike.

Carboxylic acid C-101 is also a suitable starting material for thesynthesis of carbamyls. Treatment of the acid with a chloroformate suchas isobutyl chloroformate or ethyl chloroformate in the presence of abase yields a mixed anhydride. Treatment of the mixed anhydride with anamine (such as dimethylamine, methylamine or benzylamine) yields thecarbamyl wherein R₁ and R₂ are the substituents on the various amines.

Several modifications at the C7 position are made using unsaturatedketones such as the compound of Formula C-105 (shown below in SchemeS-3) as the starting material. Sulfides are synthesized by the additionof suitable thiols under basic conditions. Examples of suitable thiolsare methyl mercaptan, ethyl mercaptan and the like. Suitable basesinclude piperidine, triethylamine and the like.

Treatment of unsaturated ketones (such as the compound of Formula C-105)with thioalkanoic acids such as thioacetic acid provides C7 thioacylcompounds such as acetylthio.

A fused C6,C7 cyclopropyl substituent may be added is by treatment ofunsaturated ketones (such as the compound of Formula C-105) withdimethylsulfoxonium methylide, which is generated by treatment oftrimethylsulfoxonium halide with a suitable base (such as sodiumhydride) in a suitable solvent.

These various synthesis schemes are illustrated in Scheme S-3 below:

Steroids bearing a C6 spirocyclopropyl ring are synthesized according tothe procedures described in Scheme S-4 below. Enones such as thecompound of Formula C-110 are first protected as a C3 enol ether bytreatment with an ortho ester such as triethyl orthoformate or trimethylorthoformate in the presence of an acid such as p-toluenesulfonic acid.The resultant enol ether is treated with Vilsmeier reagent generated insitu by addition of phosphorous oxychloride to dimethylformamide toprovide a formyl compound such as the compound of Formula C-112.Reduction of the formyl group is effected using a hydride reducingagent, such as lithium tri-tert-butoxyaluminum hydride, in a solventsuch as tetrahydrofuran. This produces an intermediate alcohol, whichupon treatment with acid, eliminates water to provide a 6-methylenecompound such as the compound of Formula C-113. Suitable acids includehydrochloric acid in an aqueous medium. Treatment of the 6-methylenecompound with diazomethane provides an intermediate pyrazoline, whichdecomposes upon heating to give a product spirocyclopropyl compound suchas the compound of Formula C-114. The protected enol ether (such as thecompound of Formula C-111) is a versatile intermediate and treatmentwith a hydride reducing agent such as sodium borohydride, followed byacid hydrolysis, provides hydroxy compounds such as the compounds ofFormulae C-115 and C-116.

These various synthesis steps are illustrated in Scheme S-4 below:

Steroids having a C6 hydroxy substituent and a C7 ester substituent maybe synthesized according to the procedures illustrated below in SchemeS-5. An ester (such as the compound of Formula C-1) is protected at theC3 carbonyl by formation of the 3,5-dienol ether (such as the compoundof Formula C-117) using an ortho ester such as triethyl orthoformate ortrimethyl orthoformate in the presence of an acid. A suitable acid isp-toluene sulfonic acid. Treatment of the enol ether with an oxidizingagent such as meta-chloroperoxybenzoic acid results in the formation ofa hydroxy compound such as the compound of Formula C-118.

Scheme S-6 illustrates the introduction of a double bond at C1-C2position of the steroid. This is carried out by treatment of the desiredsteroid (such as the compounds of Formulae C-1; C-108 and C-114), with asuitable oxidizing agent, such as dichlorodicyanobenzoquinone in asuitable solvent (such as dioxane) at temperatures ranging up to theboiling point. C1-C2 unsaturated compounds such as the compounds ofFormulae C-127, C-128 and C-129 can be prepared in accordance with thisprocedure.

Scheme S-7 illustrates the introduction of a double bond into the fusedring. A steroid (such as the compound of Formula C-114) is treated withan ortho ester (such as triethyl orthoformate or trimethyl orthoformate)in the presence of an acid catalyst (such as p-toluensulfonic acid) togive the enol ether wherein the C3 carbonyl is protected. In the case ofthe compound of Formula C-114, because the C6 position is fullysubstituted, the enol ether formed is the C2-C3 enol ether (such as thecompound of Formula C-131). Treatment of the enol ether with a strongbase such as lithium diisopropylamide at low temperature (−78° C. to−30° C.), followed by treatment with a selenating agent, such as phenylselenenyl chloride, gives the seleno derivative such as the compound ofFormula C-132. Oxidation of the seleno derivative with an oxidizingagent such as hydrogen peroxide at, for example, room temperature in thepresence of a base such as pyridine in a solvent such as methylenechloride, causes the elimination of the seleno group and introduction ofthe double bond. Hydrolysis of the enol ether gives a ketone such as thecompound of Formula C-134.

Scheme S-8 illustrates the synthesis of double bond isomers of thecompound of Formula C-1. Treatment of various spiroxane compounds, suchas those shown in Scheme S-8, with potassium acetate, trifluoraceticanhydride and trifluoroacetic acid under conditions similar to those forthe synthesis of the compound of Formula C-1 give the compounds ofFormulae C-121 and C-123.

Scheme S-9 illustrates an alternative method for the synthesis of doublebond isomers in this family of steroids. Starting with a preformed enone(such as the compound of Formula C-24 whose synthesis is describedabove) already bearing the fused ring, the C6-C7 fused cyclopropane isintroduced using the chemistry described above in Scheme S-3 for thesynthesis of compounds such as those of Formulae C-108 and C-109.

wherein X is halogen.

Fused ring steroids having an aromatic A ring can be prepared bytreating steroids such as those described in P. Compain, et al.,Tetrahedron, 52(31), 10405-10416 (1996) (which is incorporated byreference herein) with trifluoroacetic acid, potassium acetate andtrifluoroacetic anhydride in substantially the same manner as discussedabove with respect to Scheme S-1.

Those novel fused ring steroids possessing an aromatic A ring and a3-hydroxy substituent are expected to undergo all chemical reactionswhich are typical of phenols. Scheme S-10 illustrates the synthesis of a3-phenolic ether from a such a fused ring steroid. In particular,treatment of these phenolic compounds with a base and an alkyl halide oralkyl sulfonate is expected to produce the corresponding phenolic ester.See, for example, Feuer and Hooz, In The Chemistry of the Ether Linkage,Patai (Ed.), Interscience: New York, pp. 446-450, 460-468 (1967); andOlson, W. T., J.Am.Chem.Soc., 69, 2451 (1947); which are incorporatedherein by reference.

Similarly, Scheme S-11 illustrates the synthesis of a 3-phenolic esterfrom a fused ring steroid possessing an aromatic A ring and a 3-hydroxysubstituent. In particular, treatment of these phenolic compounds with acarboxylic acid anhydride or with a carboxylic acid halide is expectedto provide the corresponding phenolic ester. See, for example, March,J., Advanced Organic Chemistry, Wiley: New York, pp.346-347 (1985),which is incorporated herein by reference.

Scheme S-12 illustrates the synthesis of a 3-phenolic carbonate from afused ring steroid possessing an aromatic A ring and a 3-hydroxysubstituent. In particular, treatment of these phenolic compounds withan alkyl haloformate is expected to provide the corresponding phenoliccarbonate. See, for example, March, J., Advanced Organic Chemistry,Wiley: New York, pp.346-347 (1985), which is incorporated herein byreference.

Scheme S-13 illustrates the synthesis of ortho allyl substituted phenylderivatives from a fused ring steroid possessing an aromatic A ring anda 3-hydroxy substituent. In particular, treatment of these compoundswith a base and an allyl halide is expected to provide the correspondingallyl phenyl ether. The allyl phenyl ether should give a mixture ofortho allyl substituted phenyl derivatives upon thermal rearrangement.See, for example, Shine, H., J.Aromatic Rearrangements; ReactionMechanisms in Organic Chemistry, Monograph 6, American Elsevier: NewYork, pp. 89-120 (1967), which is incorporated herein by reference.

Scheme S-14 illustrates the synthesis of ortho dialkylated substitutedphenyl derivatives from a fused ring steroid possessing an aromatic Aring and a 3-hydroxy substituent. In particular, treatment of thesecompounds with an alcohol and an acid is expected to provide thecorresponding ortho dialkylated phenyl derivatives. See, for example,Calcott, W. S., J.Am.Chem.Soc., 61, 1010 (1939), which is incorporatedherein by reference.

Scheme S-15 illustrates the allylic oxidation of a fused ring steroidpossessing an aromatic A ring and a 3-hydroxy substituent. Inparticular, these compounds may be oxidized at an allylic position byreaction with selenium dioxide and t-butyl hydroperoxide to form thecorresponding alcohol. Dehydration of this alcohol affords thecorresponding olefin. See, for example, Schmuff, N. R., J.Org.Chem., 48,1404 (1983), which is incorporated herein by reference.

Scheme S-16 illustrates the protection of the 3-carbonyl group and thereduction of the 20-carbonyl group of the novel fused ring steroids ofthe present invention. In particular, these compounds may be reactedwith trialkylorthoformate and an acid to form the 3-enol ether. The3-enol ether can he reacted with sodium borohydride to reduce the C-20carbonyl group to the corresponding C-20 alcohol. Treatment of the C-20alcohol with an acid and water deprotects the 3-enol ether to form the3-keto derivative.

Scheme S-17 illustrates the hydrogenation of olefinic bonds in the novelfused ring steroids of the present invention. In particular,hydrogenation is expected to proceed step-wise. The C-6,C-7 double bondis first saturated followed by saturation of the C-8,C-14 double bond.

Scheme S-18 illustrates the rearrangement of protected 11α-hydroxy fusedring steroids of the present invention. In particular, the 11α-hydroxygroup of the compound is first protected with a suitable protectinggroup such as 2-methoxyethoxymethyl ether (MEM ether). Treatment of theprotected 11α-hydroxy steroid with an alkali metal salt and atrihaloalkaoic acid in the presence of an acid anhydride is expected tolead to the rearrangement of the lactone moiety in these molecules asillustrated below. Removal of the MEM ether with zinc bromide willprovide the rearranged alcohols shown below.

Scheme S-19 illustrates the protection of the 3-carbonyl group and thealkylation of the 19-position of the novel fused ring steroids of thepresent invention. In particular, the compound is first converted to the3-alkyl enol ether as illustrated in Scheme S-16. Treatment of the3-alkyl enol ether with lithium diisopropylamide (LDA) followed bytreatment with an alkyl halide leads to the formation of the 19-alkylderivative. Hydrolysis of the 3-alkyl enol ether protecting group givesthe 3-keto derivative.

Scheme S-20 illustrates the conversion of estrone methyl ether to thecorresponding spirolactone. Rearrangement of the lactone to thecorresponding fused ring steroid should occur upon treatment of thelactone with a trihalogenated alkanoic acid, preferably in the presenceof an alkali metal salt of the alkanoic acid utilized, under thereaction conditions previously disclosed. See, for example, Otsubo, K.,Tetrahedron Letters, 27(47), 5763 (1986).

The novel compounds described herein additionally may be subjected tobioconversion processes similar to those disclosed previously to yieldyet other novel fused ring steroids, such as steroids having an 9α, 9β,11α or 11β-hydroxy substituent as well as other hydroxylated fused ringsteroids. If desired, such hydroxylated steroids then can be oxidizedvia elimination of the hydroxy substituent to introduce an olefinicdouble bond such as a Δ^(9,11) olefinic double bond.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above is compositions andprocesses without departing from the scope of the invention, it isintended that all matter contained in the above description shall beinterpreted as illustrative and not in a limiting sense.

The following non-limiting examples serve to illustrate various aspectsof the present invention:

EXAMPLE X-1A Preparation of methyl2,3,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3aβ,11aβ-dimethyl-1,9-dioxo-1H-pentaleno[1,6a-a]phenanthrene-6α-carboxylate(Compound C-1)

Potassium acetate (6.7 g, 7.1 mmol.; Sigma-Aldrich 5128LG) was added toa clean, dry reactor equipped with a mechanical stirrer, condenser,thermocouple and heating mantle. Trifluoroacetic acid (25.0 mL, 8.1 mol;Sigma-Aldrich 7125MG) and trifluoroacetic anhydride (4.5 mL, 31.0 mmol;Sigma-Aldrich 11828PN) were successively added to the reactor. Thesolution was then maintained at a temperature between 25° to 30° C. for30 minutes.

The preformed TFA/TFA anhydride reagent was added to 5.0 g (9.6 mmol) of7-methyl hydrogen17-hydroxy-11α-(methylsulfonyl)oxy-3-oxo-17α-pregna-4-ene-7α,21-dicarboxylate,γ-lactone:

which was prepared in the manner described in Example 36. The resultingmixture was heated at 60° C. for 60 minutes, the degree of conversionbeing periodically checked by TLC and/or HPLC. When the reaction wascomplete (approximately 60 minutes), the mixture was transferred to1-neck flask and concentrated under reduced pressure at 50° C. until itbecame a thick slurry.

The resulting slurry was diluted with 150 mL of ethyl acetate and 80 mLof a water/brine mixture. The phases were then allowed to separate andthe aqueous layer was reextracted with 80 mL of ethyl acetate. Brinestrength was 12% by weight. The combined ethyl acetate solution waswashed once with 12% by weight brine (80 mL), then once with 1N NaOHsolution (80 mL), and finally with 12% by weight brine (80 mL). Themixture was allowed to stand for separation and the separated ethylacetate layer was concentrated to dryness under reduced pressure at 45°C. using a water aspirator to provide about 3.8 g of a crude solidproduct. HPLC analysis of the crude product revealed that the productcontained about 40 area % of compound C-1.

The solid product then was subjected to chromatographic purification Thechromatographic purification produced 210 mg of methyl2,3,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3aβ,11aβ-dimethyl-1,9-dioxo-1H-pentaleno[1,6a-a]phenantrene-6α-carboxylate(Compound C-1).

The mass spectrometry data indicated a molecular weight of 380 and aformula of C₂₄H₂₈O₄ from high resolution data. The EI mass spectrum hadan M+ peak at m/z 380. The APCI mass spectrum had peaks at m/z 381 (MH)+and m/z 398 (MNH₄)+. Carbon and hydrogen analyses were consistent withthe proposed molecular formula.

The IR spectrum had two peaks in the carbonyl absorption region: 1722cm-¹ and 1667 cm-¹. The 1722 cm-¹ peak was assigned to two carbonyls,since the ¹³C NMR spectrum had signals at δ 217.7, due to a saturatedketone, and at δ 172.7, due to carbomethoxy carbonyl. Absence of the1773 cm-¹ peak in the IR spectrum indicated loss of the lactone moiety.

The ¹³C APT and HETCOR NMR data indicated the presence of the followingtypes of carbons: 3 carbonyls (δ 217.7, 198.4, 172.2); 4 fullysubstituted olefinic carbons (δ 166.3, 141.8, 139.3, 121.8); 2 methineolefinic carbons (δ 124.9, 122.0); 3 quaternary aliphatic carbons (δ61.1, 50.7, 39.7); 1 methine aliphatic carbon (δ 43.3); 8 methylenecarbons (δ 46.0, 37.5, 34.1, 33.3, 32.9, 31.9, 23.7, 22.2); and 3 methylcarbons (δ 51.9, 23.6, 23.1).

EXAMPLE X-1B Preparation of(7α,13R,17β)-3′,4′,5′,17-tetrahydro-14-hydroxy-17-methyl-3,5′-dioxo-γ-lactone,cyclopenta[13,17]-18-norandrosta-4,9(11)-diene-7-carboxylic acid(Compound C-201)

Potassium acetate (8.9 g, 90 mmol), trifluoroacetic acid (150 mL, 1.480g/mL) and trifluoroacetic anhydride (33 mL, 1.487 g/mL) were added tothe 250 mL round bottom reactor equipped with mechanical stirrer,condenser, and heating mantel. The resulting solution was stirredbetween about 25° C. to 30° C. for about 10 minutes.

The preformed TFA/TFA anhydride reagent was added to 15 g (30.0 mmol) of7-methyl hydrogen17-hydroxy-11α-(methylsulfonyl)oxy-3-oxo-17α-pregna-4-ene-7α,21-dicarboxylate,γ-lactone

which was prepared in the manner described in Example 36. The resultingmixture was heated between about 60 to 70° C. for about 1 to 1.5 hours.This mixture was concentrated under reduced pressure at 50° C. to give athick slurry. The slurry was dissolved in 100 mL ethyl acetate and waswashed 2 times with about 20% water/brine (80 mL each time), 1 time witha 1N sodium hydroxide (80 mL) solution, followed by 1 time with about20% water/brine (80 mL). The crude product was dried over magnesiumsulfate filtered and concentrated to give about 18 g of crude wetmaterial.

This material was purified by column chromatography twice to affordabout 3 g of pure(7α,13R,17β)-3′,4′,5═,17-tetrahydro-14-hydroxy-17-methyl-3,5′-dioxo-γ-lactone,cyclopenta[13,17]-18-norandrosta-4,9(11)-diene-7-carboxylic acid(Compound C-201).

EXAMPLE X-1C Preparation of[13S,17β]-3′,4′-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-1,3,5(10)-triene-5′[2′H]-one(Compound C-202)

Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid (150 mL, 1.480g/mL) and trifluoroacetic anhydride (26 mL, 1.487 g/mL) were added to a250 mL round bottom reactor equipped with mechanical stirrer, condenser,and heating mantel. The resulting solution was stirred between about 25°C. to 30° C. for about 10 minutes.

The preformed TFA/TFA anhydride reagent was added to 15 g (43.7 mmol) of17-hydroxy-3-oxo-17α-pregn-4-ene-21-carboxylic acid, γ-lactone (alsoknown as aldonai; G. D. Searle & Co.):

The resulting mixture was heated between 60° C. to 70° C. for about 1 to1.5 hours. The reaction mixture was concentrated under reduced pressureat 50° C. to give thick slurry. The slurry was dissolved in 100 ml ethylacetate and was washed 2 times with about 20% water/brine solution (80mL each time), 1 time with 1N sodium hydroxide solution (80 mL),followed by 1 time with about 20% water/brine solution (80 mL). Thecrude product was dried over magnesium sulfate, filtered andconcentrated to dryness under reduced pressure at 50° C. to give about20 g of crude wet material.

This material was purified by column chromatography twice to affordabout 125 g of pure[13S,17β]-3′,4′-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-1,3,5(10)-triene-5′[2′H]-one(Compound C-202).

EXAMPLE X-1D Preparation of[13S,17β]-3′,4′-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-1,3,5(10),6-tetraene-5′[2′H]-one(Compound C-203)

Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid (150 mL, 1.480g/mL) and trifluoroacetic anhydride (26 mL, 1.487 g/mL) was added to a250 mL round bottom reactor equipped with mechanical stirrer, condenser,and heating mantel. The resulting solution was stirred between about 25°C. to 30° C. for about 10 minutes.

The preformed TFA/TFA anhydride reagent was added to 15 g (45.9 mmol) ofwas added to 17-hydroxy-3-oxo-17α-pregn-4,9(11)-diene-21-carboxylicacid, γ-lactone (also known as Δ-9,11-aldona):

which was prepared from 3-methoxy-3,5,9(11)-androstatriene-17-one(Upjohn). The resulting mixture was heated between about 60° C. to 70°C. for about 1 to 1.5 hours. The reaction mixture was concentrated underreduced pressure at 50° C. to give thick slurry. The slurry wasdissolved in 100 ml ethyl acetate and was washed 2 times with about 20%water/brine solution (80 mL each time), 1 time with 1N sodium hydroxidesolution (80 mL), followed by 1 time with about 20% water/brine solution(80 mL). The crude product was dried over magnesium sulfate, filteredand concentrated to dryness under reduced pressure at 50° C. to giveabout 18 g of crude wet material.

This material was purified by column chromatography twice to affordabout 340 g of pure[13S,17β]-3′,4′-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-1,3,5(10),6-tetraene-5′[2′H]-one(Compound C-203).

EXAMPLE X-1E Preparation of[13S,17β]-3′,4′-dihydro-17-methyl-cyclopenta[13,17]-18-norandrosta-4,6,8(14)-triene-3,5′[2′H]-dione(Compound C-204)

Potassium acetate (8 g, 81.5 mmol), trifluoroacetic acid (150 mL, 1.480g/mL) and trifluoroacetic anhydride (33 mL, 1.487 g/mL) were added to a250 mL round bottom reactor equipped with mechanical stirrer, condenser,and heating mantel. The resulting solution was stirred between about 25°C. to 30° C. for about 10 minutes.

The preformed TFA/TFA anhydride reagent was added to 15 g (44.0 mmol) of17-hydroxy-3-oxo-17α-pregn-4,6-diene-21-carboxylic acid, γ-lactone (alsoknown as canrenone; G. D. Searle & Co.):

The resulting mixture was heated between about 60 to 70° C. for about 1to 1.5 hours. The reaction mixture was concentrated under reducedpressure at 50° C. to give thick slurry. The slurry was dissolved in 100ml ethyl acetate and was washed 2 times with about 20% water/brinesolution (80 mL each time), 1 time with 1N sodium hydroxide solution (80mL), followed by 1 time with about 20% water/brine solution (80 mL). Thecrude product was dried over magnesium sulfate, filtered andconcentrated to dryness under reduced pressure at 50° C. to give about18 g of crude wet material.

This material was purified by column chromatography twice to affordabout 2.2 g of pure[13S,17β]-3′,4′-dihydro-17-methyl-cyclopenta[13,17]-18-norandrosta-4,6,8(14)-triene-3,5′[2′H]-dione(Compound C-204).

EXAMPLE X-2 Preparation of

To a stirred, cold (0° C.) solution of 11α-hydroxycanrenone (3.6 g, 10mmol) and triethylamine (1.2 g, 12 mmol) in methylene chloride (20 mL)is added methanesulfonyl chloride (1.1 g, 10 mmol). The mixture isstirred in the cold for 2 hours and allowed to warm to room temperature.Stirring is continued until thin layer chromatography indicates thereaction is complete. The mixture then is diluted with ethyl acetate andextracted with water, aqueous 5% sodium bicarbonate solution and waterand dried over sodium sulfate. The drying agent is filtered and thefiltrate concentrated in vacuo to give the following crude mesylateC-136 which is suitable for use in the next step:

A solution of mesylate C-136 (4.3 g, 10 mmol) is reacted withtrifluoracetic acid (25 mL), trifluoracetic anhydride (4.5 mL) andpotassium acetate (6.7 g, 7.1 mmol) according to the procedure describedfor the synthesis of Compound C-1 in Example X-1. The crude product isisolated according to the same procedure as for Compound C-1 in ExampleX-1 and is purified by chromatography on silica gel using mixtures ofethyl acetate and toluene or ethyl acetate and hexane as eluents. Theproduct thus obtained is further purified by recrystallization fromalcohol, alcohol and water, or ethyl acetate and hexane to yield totetraene C-105.

EXAMPLE X-3 Preparation of

Mesylate C-138:

is synthesized and isolated (according to the procedure described inExample X-2 for the synthesis of mesylate C-136) using11α,17-dihydroxy-3-oxo-17-oxo-pregn-4-ene-21-carboxylic acid, γ-lactone(3.6 g, 10 mmol), triethylamine (1.2 g, 12 mol) and methanesulfonylchloride (1.1 g, 10 mmol) in methylene chloride (20 mL). The mesylateC-138 thus isolated is suitable for use in the following step.

A solution of mesylate C-138 (4.4 g, 10 mmol) is reacted withtrifluoracetic acid (25 mL), trifluoracetic anhydride (4.5 mL) andpotassium acetate (6.7 g, 7.1 mmol) according to the procedure describedfor the synthesis of Compound C-1 in Example X-1. The crude productC-110 is isolated according to the same procedure as for Compound C-1 inExample X-5 and is purified by chromatography on silica gel usingmixtures of ethyl acetate and toluene or ethyl acetate and hexane aseluents. The product thus obtained is further purified byrecrystallization from alcohol, alcohol and water, or ethyl acetate andhexane.

EXAMPLE X-4 Preparation of3′,4′,5′,17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta[13R,17]-18-norandrosta-4,8,14-triene-7α-carboxylicacid (Compound C-101)

A solution of Compound C-1 (3.8 g, 10 mmol) and aqueous 1N sodiumhydroxide solution (35 mL) in ethanol (60 mL) is refluxed for 8 hours.The reaction is cooled to room temperature, concentrated on the rotaryevaporator in vacuo and the residual aqueous layer is extracted threetimes with ethyl acetate. The aqueous layer is then acidified with 1Nhydrochloric acid solution and extracted three times with ethyl acetate.The combined organic layers are washed with water and dried over issodium sulfate. The drying agent is filtered and the filtrate isconcentrated on the rotary evaporator. The residual crude carboxylicacid C-101 is crystallized by treatment with ethyl acetate and isrecrystallized from ethyl acetate and hexane or methanol or ethanol andwater.

EXAMPLE X-5 Preparation of 1-methylethyl3′,4′,5′,17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta[13R,17]-18-norandrosta-4,8,14-triene-7α-carboxylate(Compound C-102)

A mixture of sodium bicarbonate (3.5 g) and a solution of carboxylicacid C-101 (3.7 g, 10 mmol) and isopropyl iodide (3 mL) indimethylformamide (35 mL) is stirred at room temperature overnight. Thereaction is poured onto water and the aqueous solution is extractedthree times with ethyl acetate. The combined organic layers are washedwith water and dried over sodium sulfate. The drying agent is filteredand the filtrate concentrated in vacuo. The residual crude isopropylester C-102 is crystallized by treatment with ethyl acetate or alcoholand is purified by chromatography on silca gel and recrystallizationfrom ethyl acetate and hexane or alcohol or alcohol and water.

EXAMPLE X-6 Preparation of ethyl3′,4′,5′,17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta[13R,17]-1B-norandrosta-4,8,14-triene-7α-carboxylate

A mixture of sodium bicarbonate (3.5 g) and a solution of carboxylicacid C-101 (3.7 g, 10 mmol) and ethyl iodide (3 mL) in dimethylformamide(35 mL) is stirred at room temperature overnight. The reaction is pouredonto water and the aqueous solution is extracted three times with ethylacetate. The combined organic layers are washed with water and driedover sodium sulfate. The drying agent is filtered and the filtrateconcentrated in vacuo. The residual crude ethyl ester C-103 iscrystallized by treatment with ethyl acetate or alcohol and is purifiedby chromatography on silca gel and recrystallization from ethyl acetateand hexane or alcohol or alcohol and water.

EXAMPLE X-7 Preparation of hexyl3′,4′,5′,17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta[13R,17]-18-norandrosta-4,8,14-triene-7α-carboxylate(Compound C-104)

A mixture of sodium bicarbonate (3.5 g) and a solution of carboxylicacid C-101 (3.7 g, 10 mmol) and n-hexyl iodide (3 mL) indimethylformamide (35 mL) is stirred at room temperature overnight. Thereaction is poured onto water and the aqueous solution is extractedthree times with ethyl acetate. The combined organic layers are washedwith water and dried over sodium sulfate. The drying agent is filteredand the filtrate concentrated in vacuo. The residual crude n-hexyl esterC-104 is crystallized by treatment with ethyl acetate or alcohol and ispurified by chromatography on silca gel and recrystallization from ethylacetate and hexane or alcohol or alcohol and water.

EXAMPLE X-8 Preparation of3′,4′-dihydro-17-methyl-7α(methylthio)-cyclopenta[13,17]-18-norandrosta-4,8,14-triene-3,5′(2′H)-dione(Compound C-106)

A solution of tetraene C-105 (3.2 g, 10 mmol) in methanol (40 mL) andpiperidine (4 mL) is cooled to 5° C. Gaseous methyl mercaptan is passedthrough until a weight gain of 7 g is observed. The pressure containeris sealed and held at room temperature for 20 hours. The solution ispoured onto ice water and the precipitate is filtered, washed with waterand air dried. The methylthio product C-106 is purified byrecrystallization from methanol or ethyl acetate and hexane. See, e.g.,the procedure set forth in A. Karim and E. A. Brown, Steroids, 20, 41(1972), which is incorporated herein by reference.

EXAMPLE X-9 Preparation of7α-(acetylthio)-3,4′-dihydro-17-methyl-cyclopenta[13,17]-18-norandrosta-4,8,14-triene-3,5′(2′H)-dione(Compound C-107)

A solution of tetraene C-105 (3.2 g, 10 mmol) in thioacetic acid (10 mL)is heated t 85-95 C for 1 hour. Excess thioacetic acid is removed invacuo and the resultant crude 7α-thioacetate C-107 is purified byrecrystallization from a suitable solvent such as methanol or ethylacetate or ethyl acetate and hexane. See e.g., the procedure set forthin U.S. Pat. No. 3,013,012, J. A. Cella and R. C. Tweit, Dec. 12, 1961which is incorporated herein by reference.

EXAMPLE X-10 Preparation of1,2,4bR(4bR*),5,5aS*,7,7aR*,8,9,11,12bS*-dodecahydro-7a,12b-dimethyl-10aR*-cyclopropal[1]pentaleno[1,6a-a]phenanthrene-3,10-dione

1,2,4bS(4bR*),5,5aS*,8,9,11,12,12bR*-dodecahydro-7a,12b-dimethyl-10aS*-cyclopropal[1]pentaleno[1,6a-a]phenanthrene-3,10-dione(Compound C-109):

To a solution of trimethylsulfoxonium iodide (1 g, 4.6 mmol) in drydimethylsulfoxide (20 mL) is added sodium hydride (220 mg of 50%dispersion in mineral oil, 4.6 mmol). The mixture is stirred at roomtemperature under nitrogen until the evolution of hydrogen ceases. Asolution of tetraene C-105 (1.12 g, 3.5 mmol) in dimethylsulfoxide (4mL) is then added and stirring is continued for 4 hours under a nitrogenatmosphere. The reaction mixture is diluted with water and the resultantprecipitate is filtered and air dried. The product is a mixture of the6α,7β (Compound C-108) and 6α,7β (Compound C-109) isomers. Separation ofthese isomers is effected by chromatography on silica gel and theindividual isomers are further purified by recrystallization fromsolvents such as ethyl acetate and hexane, alcohol, or alcohol andwater.

EXAMPLE X-11 Preparation of

To a suspension of enone C-110 (3.2 g, 10 mmol) in ethyl orthoformate(10 mL) and anhydrous ethanol (10 mL) is added p-toluenesulfonic acidmonohydrade (0.05 g). The reaction is stirred for 30 minutes at roomtemperature and is quenched by the addition of a few drops of pyridine.After stirring another 5 minutes at 0° C., the resultant precipitate isfiltered, washed with a small amount of methanol, and recrystallizedfrom alcohol or ethyl acetate and hexane containing traces of pyridineto provide pure enol ether C-111. Alternatively, the reaction may beworked up after addition of pyridine by removing all solvents in vacuoand crystallizing the residue by addition solvents such as ether, ethylacetate or hexane. The crude C-111 is recrystallized as above. See,e.g., the procedure set forth in R. M. Weier and L. M. Hofmann, J. Med.Chem., 20, 1304-1308 (1977) which is incorporated herein by reference.

EXAMPLE X-12 Preparation of

Procedure of R. M. Weier and L. M. Hofmann, J. Med. Chem, 20 1304-1308(1977) See, e.g., which is incorporated herein by reference.

Vilsmeyer reagent is prepared by adding phosphorus oxychloride (4.59 g,30 mmol) to dimethylformamide (30 mL) at)° C. After 5 minutes, asolution of enol ether C-111 (3.5 g, 10 mmol) in dimethylformamide (5mL) is added and the reaction is stirred at 0° C. for 2 hours and atroom temperature overnight. The reaction is poured onto aqueous sodiumacetate solution and stirred for 2 hours. The precipitate is filteredand dried to give crude aldehyde C-112. Purification is effected byrecrystallization from solvents such as alcohol, alcohol and water orethyl acetate and hexane. Alternatively, the crude aldehyde C-112 isisolated from the aqueous sodium acetate solution by extraction with asolvent such as ethyl acetate. After drying over sodium sulfate andremoving the solvent, the residue is purified by recrystallization or bychromatography over silica gel followed by recrystallization.

EXAMPLE X-13 Preparation of

To a stirred cold solution of lithium tri-tert-butoxyaluminum hydride(3.05 g, 12 mmol) in tetrahydrofuran is added aldehyde C-112 (3.78 g, 10mmol). The reaction is stirred at room temperature for 5 hours andquenched by adding water and acetic acid, care being taken to keep themixture slightly basic. The mixture is concentrated in vacuo and theresultant solid slurried in ethyl acetate. The solid is filtered and thefiltrate concentrated in in vacuo. The residue is dissolved in a minimumvolume of a solution of acetone and water (3:1) and added to acidifiedaqueous acetone (pH 1.5-2.0). The acid reaction mixture is stirred atroom temperature for 1 hour and concentrated in vacuo. The resultantcrude dienone C-113 is purified by recrystallization from solvents suchas alcohol, alcohol and water or ethyl acetate and hexane.Alternatively, the crude dienone C-113 is purified by chromatography onsilica gel and then recrystallized. See, e.g., the procedure set forthin R. M. Weier and L. M. Hofmann, J.Med. Chem., 20, 1304-1308 (1977)which is incorporated herein by reference.

EXAMPLE X-14 Preparation of2′,3′,3′aα,4′,6′,10′,11′,11′aα,12′,13′-decahydro-3′aR,3′a,11′a-dimethyl-13′aR*-spiro[cyclopropane-1,7′(9′H)-[1H]pentaleno[1,6a-a]phenanthrene]-1′9′-dione

To a solution of dienone C-113 (1.17 g, 0.05 mmol) in tetrahydrofuran(30 mL) is added a solution of diazomethane (0.2 g, 0.07 mmol) in ether(7 mL). The resultant reaction solution is stored at room temperaturefor several days. Acetic acid is then added to destroy excessdiazomethane and the reaction is concentrated in vacuo. The residue,which is the intermediate pyrazoline, is crystallized under a solventsuch as acetone, hexane, ethyl acetate or ethanol and is thenrecrystallized. This material is converted to the spirocyclopropaneC-114. Thus, the solid C-114 is heated at 190° C. in vacuo and theresulting solid is recrystallized from alcohol, alcohol and water,acetone and water or ethyl acetate and hexane. Alternatively, a solutionof the pyrazoline in acetone is treated with boron trifluoride etherateat room temperature for about 1 hour. Water is added and the resultantprecipitate is filtered and air dried. Purification is effected byrecrystallization. See, e.g., the procedure set forth in F. B. Coltonand R. T. Nicholson, U.S. Pat. No. 3,499,891, Mar. 10, (1970) which isincorporated herein by reference.

EXAMPLE X-15 Preparation of

To a solution of enol ether C-111 (3.2 g, 10 mmol) in methanol (20 mL)is added sodium borohydride (38 mg). The reaction is stirred at roomtemperature for 3 hours and is treated with 1N hydrochloric acid for 30minutes. The reaction is further diluted with water and the precipitatefiltered, washed with water and dried. The product, consisting of amixture of two epimeric alcohols (Compound C-115 and Compound C-116), ischromatographed on silica gel to effect separation. The purifiedalcohols C-115 and C-116 are each recrystallized from solvents suchalcohol, alcohol and water, ethyl acetate and hexane and acetone andhexane. Alternatively, the diluted reaction mixture is extracted withethyl acetate, the combined organic layers dried over sodium sulfate andthe crude product thus isolated is chromatographed as above to providethe separated alcohols C-115 and C-116.

EXAMPLE X-16 Preparation of

Compound C-1 (3.8 g, 10 mmol) is converted to enol ether C-117 usingethyl orthoformate (3.2 g, 10 mmol), ethanol (10 mL) andp-toluenesulfonic acid monoydrate (0.05 g) according to the proceduredescribed for the synthesis of Compound C-111 set forth in Example X-11.

EXAMPLE X-17 Preparation of methyl3′,4′,5′,17-tetrahydro-6α-hydroxy-17β-methyl-3,5′-dioxocyclopenta[13R,17]-18-norandrosta-4,8,14-triene-7α-carboxylate(Compound C-118)

A solution of 57% m-chloroperoxybenzoic acid (3.64 g) in 10% aqueousdioxane (20 mL) is half-neutralized with 1N sodium hydroxide solution.This solution is cooled to 0° C. and added in portions to a stirred,cold (0° C.) solution of enol ether C-117 (4.1 g, 10 mmol) in 10%aqueous dioxane (20 mL). The reaction is stirred at room temperatureovernight, poured onto ice water and extracted with methylene chlorideor ethyl acetate. The combined organic layers are dried over sodiumsulfate. Evaporation of the solvent provides the crude hydroxy esterC-118, which is purified by chromatography on silica gel. See, e.g., theprocedure set forth in R. M. Weier and L. M. Hofmann, J.Med.Chem., 20,1304-1308 (1977) which is incorporated herein by reference.

EXAMPLE X-18 Preparation of

Lithium dimethyl cuprate is prepared by adding methyllithium (19 mL of a1.6 M solution in ether, 30 mmol) to a stirred suspension of cuprousiodide (2.86 g, 15 mmol) in ether (30 mL) at 0° C. under an inertatmosphere of argon. After stirring in the cold for 15 minutes asolution of tetraene C-105 (1.6 g, 15 mmol) in tetrahydrofuran (25 mL)is added dropwise over 25 minutes. The reaction is continued for anadditional 30 minutes and poured onto saturated ammonium chloridesolution with vigorous stirring. The aqueous mixture is extracted withethyl acetate or methylene chloride. The combined organic layers arewashed with aqueous ammonium chloride solution, water and dried oversodium sulfate. The solvent is removed in vacuo and the residue isdissolved in ethyl acetate or methylene chloride and treated withp-toluenesulfonic acid (100 mg) on the steam bath for 30 minutes to 1hour. The organic solution is washed with water and dried over sodiumsulfate. The solvent is removed in vacuo to give the crude enone C-119.Purification is effected by recrystallization from alcohol, alcohol andwater or ethyl acetate and hexane. Alternatively, the crude enone C-119is chromatographed on silica gel and the product then recrystallized.See, e.g., the procedure set forth in J. K. Grunwell et al., Steroids,27, 759-771 (1976), which is incorporated herein by reference.

EXAMPLE X-19 Preparation of

Ester C-120 (4.00 g, 10 mmol):

(prepared in accordance with the procedure set forth in R. M. Weier andL. M. Hofmann, J.Med.Chem., 18, 817 (1975), which is incorporated hereinby reference) is reacted with trifluoroacetic acid (25 mL),trifluoroacetic anhydride (4.5 mL) and potassium acetate (6.7 g, 7.1mmol) according to the procedure described for the synthesis of CompoundC-1 in Example X-1. The crude product C-121 is isolated according to thesame procedure as for Compound C-1 in Example X-1 and is purified bychromatography on silica gel using mixtures of ethyl acetate and tolueneor ethyl acetate and hexane as eluents and the product thus obtained isfurther purified by recrystallization from alcohol, alcohol and water,or ethyl acetate and hexane.

EXAMPLE X-20 Preparation of

Enone C-122 (3.68 g, 10 mmol).

(prepared in accordance with the procedure set forth in F. B. Colton andR. T. Nicholson, U.S. Pat. No. 3,499,891, Mar. 10, 1970, which isincorporated herein by reference) is reacted with potassium acetate (6.7g, 7.1 mmol), trifluoracetic acid (25 mL) and trifluoroacetic acidanhydride (4.5 mL) according to the procedure described for thesynthesis of Compound C-1 in Example X-1. The crude product is isolatedas described in the above reference and is purified by chromatography onsilica gel using mixtures of ethyl acetate and hexane or ethyl acetateand toluene as eluents. The purified C-123 is further purified byrecrystallization from alcohol, alcohol and water, or ethyl acetate andhexane.

EXAMPLE X-21 Preparation of

Compounds C-125 and C-126 are synthesized according to the procedureused above for the synthesis of Compounds C-108 and C-1. Thus, to asolution of trimethylsulfoxonium iodide (1 g, 4.6 mmol) in drydimethylsulfoxide (20 mL) is added sodium hydride (220 mg of 50%dispersion in mineral oil, 4.6 mmol). The mixture is stirred at roomtemperature under nitrogen until the evolution of hydrogen ceases.

A solution of trienone C-124 (1.14 g, 3.5 mmol):

in dimethylsulfoxide (4 mL) is then added and stirring is continued for4 hours under a nitrogen atmosphere. Trienone C-124 is prepared inaccordance with the procedure set forth in Example X-2 for thepreparation of Tetraene C-105 by using canrenone as the startingmaterial instead of 11α-hydroxy canrenone. The reaction mixture isdiluted with water and the resultant precipitate is filtered and airdried. The product is a mixture of the 6α,7β (Compound C-125) and 6α,7β(Compound C-126) isomers. Separation of these isomers is effected bychromatography on silica gel and the individual isomers are furtherpurified by recrystallization from solvents such as ethyl acetate andhexane, alcohol or alcohol and water.

EXAMPLE X-22 Preparation of methyl3′,4′,5′,17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta[13R,17]-18-norandrosta-1,4,8,14-tetraene-7α-carboxylate(Compound C-127)

Dienone C-127 is synthesized according to the procedure described in R.M. Weier and L. M. Hofmann, J.Med. Chem. 18, 817(1975) which isincorporated by reference herein. Thus, a solution of Compound C-1 (3.8g, 10 mmol) and dichlorodicyanobenzoquinone (2.72 g, 12 mmol) in dioxane(80 mL) is refluxed with stirring for 24 hours. The reaction mixture isconcentrated in vacuo, the residue digested with methylene chloride,filtered and the filtrate washed with a 2% sodium sulfite, 5% sodiumhydroxide and saturated sodium chloride solution and dried over sodiumsulfate. The drying agent is filtered and the filtrate is concentratedin vacuo. The crude product dienone C-127 is purified by chromatographyon silica gel using mixtures of ethyl acetate and toluene as eluents andthe product 27 thus isolated is further purified by recrystallizationfrom alcohol.

EXAMPLE X-25 Preparation of2′,3′,3′aα,4′,6′11′aα,12′,13′-octahydro-3′aR,3′a,11′a-dimethyl-13′aR*-spiro[cyclopropane-1,7′(9′H)-[1H]pentaleno,[1,6a-a]phenanthrene]1′9′-dione(Compound C-128)

Using the procedure and workup described above in Example X-22 for thesynthesis of dienone C-127, enone C-114 (3.48 g, 10 mmol) is convertedto dienone C-128 using dichlorodicyanobenzoquione (2.72 g, 12 mmol) indioxane (80 mL).

EXAMPLE X-24 Preparation of4bR(4bR*),5,5aS*,7,7aR*,8,9,11,12,12bS*-decahydro-7A,12b-dimethyl-1OR*-cyclopropa(1)pentaleno[1,6a-a]phenanthrene-3,10-dione (Compound C-129)

Using the procedure and workup described above in Example X-22 for thesynthesis of dienone C-127, enone C-108 (3.34 g, 10 mmol) is convertedto dienone C-129 using dichlorodicyanobenzoquione (2.72 g, 12 mmol) indioxane (80 mL).

EXAMPLE X-25 Preparation of

To a cold (0° C.), stirred solution of carboxylic acid C-101 (3.66 g, 10mmol) and N-methylmorpholine (1.01 g, 10 mmol) in tetrahydrofuran (35mL) is added isobutyl chloroformate (1.36 g, 10 mmol). The reaction isstirred at 0° C. for 20 minutes, filtered and the filtrate isconcentrated in vacuo. The residue is the mixed anhydride and issuitable for use in the next step.

Gaseous dimethylamine is bubbled through a cold (0° C.) solution ofmixed anhydride (4.6 g, 10 mmol) in tetrahydrofuran (40 mL) in apressure vessel. After 15 minutes, the pressure vessel is sealed and thereaction is allowed to stand at room temperature for 24 hour. Thereaction is warmed to 40 C for 30 minutes and cooled back to 0° C. Afterwarming to room temperature, the vessel is vented to the atmosphere andexcess dimethylamine is permitted to evaporate. The reaction isconcentrated in vacuo, the residue dissolved in ethyl acetate andextracted with 1N sodium hydroxide solution and water. After drying oversodium sulfate, the organic layer is stripped and the residue purifiedby chromatography on silica gel using mixtures of ethyl acetate andtoluene as the eluents. The amide C-130 thus isolated is furtherpurified by recrystallization from alcohol, alcohol and water or ethylacetate and hexane.

EXAMPLE X-26 Preparation of

To a suspension of enone C-114 (3.5 g, 10 mmol) in ethyl orthoformate(10 mL) and anhydrous ethanol (10 mL) is added p-toluenesulfonic acidmonohydrade (0.05 g). The reaction is stirred for 30 minutes at roomtemperature and is quenched by the addition of a few drops of pyridine.After stirring another 5 minutes at 0° C., the resultant precipitate isfiltered, washed with a small amount of methanol, and recrystallizedfrom alcohol or ethyl acetate and hexane containing traces of pyridineto provide pure enol ether C-131. Alternatively, the reaction may beworked up after addition of pyridine by removing all solvents in vacuoand crystallizing the residue by addition solvents such as hexane,ether, ethyl acetate or hexane. The crude enol ether C-131 isrecrystallized as above.

EXAMPLE X-27 Preparation of

To a cold (−78° C.) solution of lithium bis(trimethylsilyl)amide (10 mLof a 1.0 M solution in tetrahydrofuran) a solution of enol ether C-131(3.8 g, 10 mmol) in tetrahydrofuran (20 mL) is added dropwise over 20minutes The reaction is stirred at −78° C. for 10 minutes and a solutionof phenylselenenyl chloride (1.9 g, 10 mmol) in tetrahydrofuran (5 mL)is added. The reaction is stirred 5 minutes and quenched by the additionof 1% sodium bisulfate solution. The reaction is further diluted withwater and extracted with ethyl acetate. The combined organic layers arewashed with 5% aqueous sodium bicarbonate solution, and water, and driedover sodium sulfate. The drying agent is filtered and the filtrateconcentrated in vacuo. The residue is purified by chromatography onsilica gel using mixtures of ethyl acetate and toluene or ethyl acetateand hexane as eluents to give pure selenide C-132.

EXAMPLE X-28 Preparation of

To a cold (0° C.) solution of selenide C-132 (g, 10 mol) and pyridine(1.61 mL, 20 mmol) in methylene chloride (40 mL) is gradually added asolution of hydrogen peroxide (3.1 g of 30% solution, 27 mmol) in water(3 mL). The temperature is maintained at less than 30-35° C. After anyexothermicityi subsides, the ice bath is removed and the reaction isstirred vigorously at room temperature for 15 minutes. The reaction isdiluted with methylene chloride and washed with 5% sodium bicarbonatesolution and water and dried over sodium sulfate. The drying agent isfiltered and the filtrate is concentrated in vacuo. The residue ispurified by chromatography on silica gel using mixtures of ethyl acetateand toluene or ethyl acetate and hexane as eluents to give pureunsaturated ketone C-133 which is further purified by recrystallizationfrom ethyl acetate and hexane or alcohol.

EXAMPLE X-29 Preparation of

A solution of ketone C-133 (2 g) in acetone (15 mL) is treated with 1Nhydrochloric acid (4 mL) for 1 hour at room temperature. The reaction isconcentrated in vacuo. The residue is diluted with water and extractedwith ethyl acetate. The combined organic layers are washed with 5%sodium bicarbonate solution and water and dried over sodium sulfate. Thedrying agent is filtered and the filtrate concentrated in vacuo to givecrude ketone C-134. The crude product is purified by chromatography onsilica gel using mixtures of ethyl acetate and toluene as eluents togive pure ketone C-134 which is further purified by recrystallizationfrom ethyl acetate and hexane or alcohol.

What is claimed is:
 1. A process for the preparation of a3-keto-7α-alkoxycarbonyl substituted Δ-4,5-steroid comprising reactingan alkylating reagent with a 4,5-dihydro-5,7-lactone steroid substratein the presence of a base, said lactone substrate being substituted withketo or dialkoxy at the 3-carbon, and further comprising the moiety:

where C(5) represents the 5-carbon and C(7) represents the 7-carbon ofthe steroid structure of the substrate.
 2. A process as set forth inclaim 1 wherein said steroid substrate comprises a saturated orunsaturated nuclear structure of Formula XLI

or of Formula XLII wherein —A—A— represents the group —CHR₄—CHR₅— or—CR₄═CR₅—; R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy; —B—B—represents the group —CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;—E—E— is selected from among:

where R²¹, R²² and R²³ are independently selected from among hydrogen,alkyl, halo, nitro, and cyano; and R²⁴ is selected from among hydrogenand lower alkyl; wherein R¹⁸ is C₁-C₄ alkyl, or the R¹⁸O— groupstogether form an O,O-oxyalkylene bridge; and wherein R⁸⁰ and R⁹⁰ areindependently selected from R⁸ and R⁹, respectively, or R⁸⁰ and R⁹⁰together form keto; where R⁸ and R⁹ are independently selected from thegroup consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl,hydroxyalkyl, alkoxyalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl,acyloxyalkyl, cyano and aryloxy, or R⁸ and R⁹ together comprise acarbocyclic or heterocyclic ring structure, or R⁸ or R⁹ together with R⁶or R⁷ comprise a carbocyclic or heterocyclic ring structure fused to thepentacyclic D ring.
 3. A process as set forth in claim 2 wherein saidsteroid substrate and said steroid product are substituted at the17-position with a spirolactone substituent corresponding to FormulaXXXIII


4. A process as set forth in claim 1 comprising a 17-keto structure. 5.A process as set forth in claim 1 wherein said lactone substratecomprises 3-dialkoxy substituents.
 6. A process as set forth in claim 1wherein said 5,7-lactone is reacted with an alkyl halide in the presenceof a base.
 7. A process for the preparation of a compound correspondingto Formula II:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—; R¹represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical; R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy; and —B—B—represents the group —CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;and R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonylalkyl, alkoxycarbonylalkyl, acyloxyalkyl, cyano andaryloxy, or R⁸ and R⁹ together comprise a carbocyclic or heterocyclicring structure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise acarbocyclic or heterocyclic ring structure fused to the pentacyclic Dring; the process comprising: alkylating a compound of Formula EI byhydrolysis followed by reaction with an alkylating reagent in thepresence of a base, the compound of Formula El having the structure:

wherein —A—A—, R³, R⁸, R⁹ and —B—B— are as defined above, and R¹⁷ is C₁to C₄ alkyl.
 8. A process for the preparation of a compoundcorresponding to Formula IIC:

wherein —A—A— represents the group —CHR₄—CHR₅— or —CR₄═CR₅—; R¹represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical; R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy; and —B—B—represents the group —CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;the process comprising: alkylating a compound of Formula E by hydrolysisfollowed by reaction with an alkylating reagent in the presence of abase, the compound of Formula E having the structure:

wherein —A—A—, —B—B— and R³, are as defined above, and R¹⁷ is C₁ to C₄alkyl.
 9. A process for the preparation of a compound corresponding toFormula I:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—; R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano and aryloxy; R²⁶ is C₁ to C₄ alkyl; and —B—B—represents the group —CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;and R⁸⁰ and R⁹⁰ are independently selected from R⁸ and R⁹, respectively,or R⁸⁰ and R⁹⁰ together form keto; R⁸ and R⁹ are independently selectedfrom the group consisting of hydrogen, hydroxy, halo, lower alkoxy,acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonylalkyl,alkoxycarbonylalkyl, acyloxyalkyl, cyano and aryloxy, or R⁸ and R⁹together comprise a carbocyclic or heterocyclic ring structure, or R⁹together with R⁶ or R⁷ comprise a carbocyclic or heterocyclic ringstructure fused to the pentacyclic D ring; the process comprising:alkylating a compound of Formula A211 by reaction with an alkylatingreagent in the presence of a base, the compound of Formula A211 havingthe structure:

wherein —A—A—, —B—B—, R³, R⁸⁰ and R⁹⁰ are as defined above.
 10. Aprocess for the preparation of a compound corresponding to Formula IE:

wherein —A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—; R³, R⁴ andR⁵ are independently selected from the group consisting of hydrogen,halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, cyano and aryloxy; R²⁶ is C₁ to C₄ alkyl; and —B—B—represents the group —CHR⁶—CHR⁷— or an alpha- or beta-oriented group:

where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy;the process comprising: alkylating a compound of Formula 211 by reactionwith an alkylating reagent in the presence of a base, the compound ofFormula 211 having the structure:

wherein —A—A—, —B—B— and R³ are as defined above.
 11. A processaccording claim 7 wherein R³ is hydrogen, —A—A— represents the group—CHR⁴—CHR⁵—, and —B—B— represents the group —CHR⁶—CHR⁷—.
 12. A processaccording to claim 8 wherein R³ is hydrogen, —A—A— represents the group—CHR⁴—CHR⁵—, and —B—B— represents the group —CHR⁶—CHR⁷—.
 13. A processaccording to claim 9 wherein R³ is hydrogen, —A—A— represents the group—CHR⁴—CHR⁵—, and —B—B— represents the group —CHR⁶—CHR⁷—.
 14. A processaccording to claim 10 wherein R³ is hydrogen, —A—A— represents the group—CHR⁴—CHR⁵—, and —B—B— represent the group —CHR⁶—CHR⁷—.
 15. A processaccording to claim 11 wherein R⁴, R⁵, R⁶ and R⁷ are each hydrogen.
 16. Aprocess according to claim 12 wherein R⁴, R⁵, R⁶ and R⁷ are eachhydrogen.
 17. A process according to claim 13 wherein R⁴, R⁵, R⁶ and R⁷are each hydrogen.
 18. A process according to claim 14 wherein R⁴, R⁵,R⁶ and R⁷ are each hydrogen.